Compositions and methods for treating or preventing diseases of body passageways

ABSTRACT

The present invention provides methods for treating or preventing diseases associated with body passageways, comprising the step of delivering to an external portion of the body passageway a therapeutic agent. Representative examples of therapeutic agents include anti-angiogenic factors, anti-proliferative agents, anti-inflammatory agents, and antibiotics.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of co-pending U.S. patent applicationSer. No. 10/671,327, filed Sep. 25, 2003, which is a continuation ofU.S. patent application Ser. No. 09/933,652, filed Aug. 20, 2001, issuedon Jul. 6, 2004, as U.S. Pat. No. 6,759,431, which is a continuation ofU.S. patent application Ser. No. 08/653,207, filed May 24, 1996, nowabandoned, which applications are incorporated herein by reference intheir entirety.

TECHNICAL FIELD

The present invention relates generally to compositions and methods fortreating or preventing diseases of body passageways, and morespecifically, to compositions comprising therapeutic agents which may bedelivered to the external walls of body passageways.

BACKGROUND OF THE INVENTION

There are many passageways within the body which allow the flow ofessential materials. These include, for example, arteries and veins, theesophagus, stomach, small and large intestine, biliary tract, ureter,bladder, urethra, nasal passageways, trachea and other airways, and themale and female reproductive tract. Injury, various surgical procedures,or disease can result in the narrowing, weakening and/or obstruction ofsuch body passageways, resulting in serious complications and/or evendeath.

For example, many types of tumors (both benign and malignant) can resultin damage to the wall of a body passageway or obstruction of the lumen,thereby slowing or preventing the flow of materials through thepassageway. In 1996 alone, it has been estimated that over 11,200 deathswill occurr due to esophageal cancer, over 51,000 deaths due to largeand small intestine cancer and nearly 17,000 deaths due to rectal cancerin the United States. Obstruction in body passageways that are affectedby cancer are not only in and of themselves life-threatening, they alsolimit the quality of a patient's life.

The primary treatment for the majority of tumors which cause neoplasticobstruction is surgical removal and/or chemotherapy, radiation therapyor laser therapy. Unfortunately, by the time a tumor causes anobstruction in a body passageway it is frequently inoperable andgenerally will not responded to traditional therapies. One approach tothis problem has been the insertion of endoluminal stents. Briefly,stents are devices placed into the lumen of a body passageway tophysically hold open a passageway that has been blocked by a tumor orother tissues/substances. Representative examples of commonly deployedstents include the Wallstent, Stecker stent, Gianturco stent and Palmazstent (see e.g., U.S. Pat. Nos. 5,102,417, 5,195,984, 5,176,626,5,147,370, 5,141,516, 4,776,337). A significant drawback however to theuse of stents in neoplastic obstruction is that the tumor is often ableto grow into the lumen through the interstices of the stent. Inaddition, the presence of a stent in the lumen can induce the ingrowthof reactive or inflammatory tissue (e.g., blood vessels, fibroblasts andwhite blood cells) onto the surface of the stent. If this ingrowth(composed of tumor cells and/or inflammatory cells) reaches the innersurface of the stent and compromises the lumen, the result isre-blockage of the body passageway which the stent was inserted tocorrect.

Other diseases, which although not neoplastic nevertheless involveproliferation, can likewise obstruct body passageways. For example,narrowing of the prostatic urethra due to benign prostatic hyperplasiais a serious problem affecting 60% of all men over the age of 60 yearsof age and 100% of all men over the age of 80 years of age. Presentpharmacological treatments, such as 5-alphareductase inhibitors (e.g.,Finasteride), or alpha-adrenergic blockers (e.g., Terazozan) aregenerally only effective in a limited population of patients.

Moreover, of the surgical procedures that can be performed (e.g.,trans-urethral resection of the prostate (TURPs); open prostatectomy, orendo-urologic procedures such as laser prostatectomy, use of microwaves,hypothermia, cryosurgery or stenting), numerous complications such asbleeding, infection, incontinence, impotence, and recurrent disease,typically result.

In addition to neoplastic or proliferative diseases, other diseases suchvascular disease can result in the narrowing, weakening and/orobstruction of body passageways. According to 1993 estimates(source—U.S. Heart and Stroke Foundation homepage), over 60 millionAmericans have one or more forms of cardiovascular disease. Thesediseases claimed 954,138 lives in the same year (41% of all deaths inthe United States).

Balloon angioplasty (with or without stenting) is one of the most widelyused treatments for vascular disease; other options such as laserangioplasty are also available. While this is the treatment of choice inmany cases of severe narrowing of the vasculature, about one-third ofpatients undergoing balloon angioplasty (source Heart and StokeFoundation homepage) have renewed narrowing of the treated arteries(restenosis) within 6 months of the initial procedure; often seriousenough to necessitate further interventions.

Such vascular diseases (including for example, restenosis) are due atleast in part to intimal thickening secondary to vascular smooth musclecell (VSMC) migration, VSMC proliferation, and extra-cellular matrixdeposition. Briefly, vascular endothelium acts as a nonthrombogenicsurface over which blood can flow smoothly and as a barrier whichseparates the blood components from the tissues comprising the vesselwall. Endothelial cells also release heparin sulphate, prostacyclin,EDRF and other factors that inhibit platelet and white cell adhesion,VSMC contraction, VSMC migration and VSMC proliferation. Any loss ordamage to the endothelium, such as occurs during balloon angioplasty,atherectomy, or stent insertion, can result in platelet adhesion,platelet aggregation and thrombus formation. Activated platelets canrelease substances that produce vasoconstriction (serotonin andthromboxane) and/or promote VSMC migration and proliferation (PDGF,epidermal growth factor, TGF-β, and heparinase). Tissue factors releasedby the arteries stimulates clot formation resulting in a fibrin matrixinto which smooth muscle cells can migrate and proliferate.

This cascade of events leads to the transformation of vascular smoothmuscle cells from a contractile to a secretory phenotype. Angioplastyinduced cell lysis and matrix destruction results in local release ofbasic fibroblast growth factor (bFGF) which in turn stimulates VSMCproliferation directly and indirectly through the induction of PDGFproduction. In addition to PDGF and bFGF, VSMC proliferation is alsostimulated by platelet released EGF and insulin growth factor −1.

Vascular smooth muscle cells are also induced to migrate into the mediaand intima of the vessel. This is enabled by release and activation ofmatrix metalloproteases which degrade a pathway for the VSMC through theextra-cellular matrix and internal elastic lamina of the vessel wall.After migration and proliferation the vascular smooth muscle cells thendeposit an extra-cellular matrix consisting of gylcosaminoglycans,elastin and collagen which comprises the largest part of intimalthickening. A significant portion of the restenosis process may be dueto remodeling of the vascular wall leading to changes in the overallsize of the artery; at least some of which is secondary to proliferationwithin the adventitia (in addition to the media). The net result ofthese processes is a recurrence of the narrowing of the vascular wallwhich is often severe enough to require a repeat intervention.

In summary, virtually any forceful manipulation within the lumen of ablood vessel will damage or denude its endothelial lining. Thus,treatment options for vascular diseases themselves and for restenosisfollowing therapeutic interventions continue to be major problems withrespect to longterm outcomes for such conditions.

In addition to neoplastic obstructions and vascular disease, there arealso a number of acute and chronic inflammatory diseases which result inobstructions of body passages. These include, for example, vasculitis,gastrointestinal tract diseases (e.g. Crohn's disease, ulcerativecolitis) and respiratory tract diseases (e.g. asthma, chronicobstructive pulmonary disease).

Each of these diseases can be treated, to varying degrees of success,with medications such as anti-inflammatories or immunosuppressants.Current regimens however are often ineffective at slowing theprogression of disease, and can result in systemic toxicity andundesirable side effects. Surgcal procedures can also be utilizedinstead of or in addition to medication regimens. Such surgicalprocedures however have a high rate of local recurrence to due to scarformation, and can under certain conditions (e.g., through the use ofballoon catheters), result in benign reactive overgrowth.

Other diseases that can also obstruct body passageways includeinfectious diseases. Briefly, there are a number of acute and chronicinfectious processes that can result in the obstruction of bodypassageways including for example, urethritis, prostatitis and otherdiseases of the male reproductive tract, various diseases of the femalereproductive tract, cystitis and urethritis (diseases of the urinarytract), chronic bronchitis, tuberculosis and other mycobacteriainfections and other respiratory problems and certain cardiovasculardiseases.

Such diseases are presently treated either by a a variety of differenttherapeutic regimens and/or by surgical procedures. As above however,such therapeutic regimens have the difficulty of associated systemictoxicity that can result in undesired side effects. In addition, asdiscussed above surgical procedures can result in local recurrence dueto scar formation, and in certain procedures (e.g., insertion ofcommercially available stents), may result in benign reactiveovergrowth.

The existing treatments for the above diseases and conditions for themost part share the same limitations. The use of therapeutic agents havenot resulted in the reversal of these conditions and whenever anintervention is used to treat the conditions, there is a risk to thepatient as a result of the body's response to the intervention. Thepresent invention provides compositions and methods suitable fortreating the conditions and diseases which are generally discussedabove. These compositions and methods address the problems associatedwith the existing procedures, offer significant advantages when comparedto existing procedures, and in addition, provide other, relatedadvantages.

SUMMARY OF THE INVENTION

Briefly stated, the present invention provides methods for treating orpreventing diseases associated with body passageways, comprising thestep of delivering to an external portion of the body passageway atherapeutic agent. Within a related aspect, methods for treating orpreventing diseases associated with body passageways are providedcomprising the step of delivering to smooth muscle cells of said bodypassageway, via the adventia, a therapeutic agent. By delivering thetherapeutic compound locally to the site of disease, systemic andunwanted side effects can be avoided and total dosages can potentiallybe reduced. Delivery quadrantically or circumferentially around diseasedpassageway also avoids many of the disadvantages of endoluminalmanipulation, including damage to the epithelial lining of the tissue.For example damage to the endothelium can result in thrombosis, changesto laminar flow patterns, and/or a foreign body reaction to anendoluminal device, any of which can initiate the restenosis cascade. Inthe case of prostatic disease, avoiding instrumentation of the urethracan reduce the likelihood of strictures and preserve continence andpotency.

A wide variety of therapeutic agents may be utilized within the scope ofthe present invention, including for example anti-angiogenic agents,anti-proliferative agents, anti-inflammatory agents, and antibiotics.

Within certain embodiments of the invention, the therapeutic agents mayfurther comprise a carrier (either polymeric or non-polymeric), such as,for example, poly(ethylene-vinyl acetate) (40% crosslinked), copolymersof lactic acid and glycolic acid, poly (caprolactone), poly (lacticacid), copolymers of poly (lactic acid) and poly (caprolactone),gelatin, hyaluronic acid, collagen matrices, and albumen.

The therapeutic agents may be utilized to treat or prevent a widevariety of diseases, including for example, vascular diseases,neoplastic obstructions, inflammatory diseases and infectious diseases.Representative body passageways which may be treated include, forexample, arteries, the esophagus, the stomach, the duodenum, the smallintestine, the large intestine, biliary tracts, the ureter, the bladder,the urethra, lacrimal ducts, the trachea, bronchi, bronchioles, nasalairways, eustachian tubes, the external auditory canal, uterus andfallopian tubes.

Within one particularly preferred embodiment of the invention, thetherapeutic agent is delivered to an artery by direct injection via anouter wall of the artery into the adventia.

These and other aspects of the present invention will become evidentupon reference to the following detailed description and attacheddrawings. In addition, various references are set forth below whichdescribe in more detail certain procedures, devices or compositions, andare therefore incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph which shows the effect of plasma opsonization ofpolymeric microspheres on the chemiluminescence response of neutrophils(20 mg/ml microspheres in 0.5 ml of cells (conc. 5×10⁶ cells/ml) to PCLmicrospheres.

FIG. 2 is a graph which shows the effect of precoating plasma+/−2%pluronic F127 on the chemiluminescence response of neutrophils (5×10⁶cells/ml) to PCL microspheres

FIG. 3 is a graph which shows the effect of precoating plasma+/−2%pluronic F127 on the chemiluminescence response of neutrophils (5×10⁶cells/ml) to PMMA microspheres

FIG. 4 is a graph which shows the effect of precoating plasma+/−2%pluronic F127 on the chemiluminescence response of neutrophils (5×10⁶cells/ml) to PLA microspheres

FIG. 5 is a graph which shows the effect of precoating plasma+/−2%pluronic F127 on the chemiluminescence response of neutrophils (5×10⁶cells/ml) to EVA:PLA microspheres

FIG. 6 is a graph which shows the effect of precoating IgG (2 mg/ml), or2% pluronic F127 then IgG (2 mg/ml) on the chemiluminescence response ofneutrophils to PCL microspheres.

FIG. 7 is a graph which shows the effect of precoating IgG (2 mg/ml), or2% pluronic F127 then IgG (2 mg/ml) on the chemiluminescence response ofneutrophils to PMMA microspheres.

FIG. 8 is a graph which shows the effect of precoating IgG (2 mg/ml), or2% pluronic F127 then IgG (2 mg/ml) on the chemiluminescence response ofneutrophils to PVA microspheres.

FIG. 9 is a graph which shows the effect of precoating IgG (2 mg/ml), or2% pluronic F127 then IgG (2 mg/ml) on the chemiluminescence response ofneutrophils to EVA:PLA microspheres.

FIG. 10A is a graph which shows the effect of the EVA:PLA polymer blendratio upon aggregation of microspheres. FIG. 10B is a scanning electronmicrograph which shows the size of “small” microspheres. FIG. 10C (whichincludes a magnified inset—labelled “10C-inset”) is a scanning electronmicrograph which shows the size of “large” microspheres. FIG. 10D is agraph which depicts the time course of in vitro paclitaxel release from0.6% w/v paclitaxel-loaded 50:50 EVA:PLA polymer blend microspheres intophosphate buffered saline (pH 7.4) at 37° C. Open circles are “small”sized microspheres, and closed circles are “large” sized microspheres.FIG. 10E is a photograph of a CAM which shows the results of paclitaxelrelease by microspheres (“MS”). FIG. 10F is a photograph similar to thatof 10E at increased magnification.

FIG. 11A is a graph which shows release rate profiles frompolycaprolactone microspheres containing 1%, 2%, 5% or 10% paclitaxelinto phosphate buffered saline at 37° C. FIG. 11B is a photograph whichshows a CAM treated with control microspheres. FIG. 11C is a photographwhich shows a CAM treated with 5% paclitaxel loaded microspheres.

FIGS. 12A and 12B, respectively, are two graphs which show the releaseof paclitaxel from EVA films, and the percent paclitaxel remaining inthose same films over time. FIG. 12C is a graph which shows the swellingof EVA/F127 films with no paclitaxel over time. FIG. 12D is a graphwhich shows the swelling of EVA/Span 80 films with no paclitaxel overtime. FIG. 12E is a graph which depicts a stress vs. strain curve forvarious EVA/F127 blends.

FIGS. 13A and 13B are two graphs which show the melting point ofPCL/MePEG polymer blends as a function of % MePEG in the formulation(13A), and the percent increase in time needed for PCL paste at 60° C.to being to solidify as a function of the amount of MePEG in theformulation (13B). FIG. 13C is a graph which depicts the softness ofvarying PCL/MePEG polymer blends. FIG. 13D is a graph which shows thepercent weight change over time for polymer blends of various MePEGconcentrations. FIG. 13E is a graph which depicts the rate of paclitaxelrelease over time from various polymer blends loaded with 1% paclitaxel.FIGS. 13F and 13G are graphs which depict the effect of varyingquantities of paclitaxel on the total amount of paclitaxel released froma 20% MePEG/PCL blend. FIG. 13H is a graph which depicts the effect ofMePEG on the tensile strength of a MePEG/PCL polymer.

FIG. 14 is a graph which shows paclitaxel release from various polymericformulations.

FIG. 15 is a graph which depicts, over a time course the release ofpaclitaxel from PCL pastes into PBS at 37° C. The PCL pastes containmicroparticles of paclitaxel and various additives prepared using mesh#140. The error bars represent the standard deviation of 3 samples.

FIG. 16 is a graph which depicts time courses of paclitaxel release frompaclitaxel-gelatin-PCL pastes into PBS at 37° C. This graph shows theeffects of gelatin concentration (mesh #140) and the size ofpaclitaxel-gelatin (1:1) microparticles prepared using mesh #140 or mesh#60. The error bars represent the standard deviation of 3 samples.

FIGS. 17A and 17B are graphs which depict the effect of additives (17A;mesh #140) and the size of microparticles (17B; mesh #140 or #60) andthe proportion of the additive (mesh #140) on the swelling behavior ofPCL pastes containing 20% paclitaxel following suspension in distilledwater at 37° C. Measurements for the paste prepared with 270 μmmicroparticles in paclitaxel-gelatin and paste containing 30% gelatinwere discontinued after 4 hours due to disintegration of the matrix. Theerror bars represent the standard deviation of 3 samples.

FIGS. 18A, 18B, 18C and 18D are representative scanning electronmicrographs of paclitaxel-gelatin-PCL (20:20:60) pastes before (18A) andafter (18B) suspending in distilled water at 37° C. for 6 hours.Micrographs 18C and 18D are higher magnifications of 18B, showingintimate association of paclitaxel (rod shaped) and gelatin matrix.

FIGS. 19A and 19B are representative photomicrographs of CAMs treatedwith gelatin-PCL (19A) and paclitaxel-gelatin-PCL (20:20:60; 19B) pastesshowing zones of avascularity in the paclitaxel treated CAM.

FIG. 20 is a table which shows the effect of peri-tumoral injection ofpaclitaxel-gelatin-PCL paste into mice with established tumors.

FIG. 21 is a table which shows the melting temperature, enthalpy,molecular weight, polydispersity and intrinsic viscosity ofPDLLA-PEG-PDLLA compositions.

FIG. 22 is a graph which depicts DSC thermograms of PDLLA-PEG-PDLLA andPEG. The heating rate was 110° C./min. See FIG. 21 for meltingtemperatures and enthalpies.

FIG. 23 is a graph which depicts the cumulative release of paclitaxelfrom 20% paclitaxel loaded PDLLA-PEG-PDLLA cylinders into PBS albuminbuffer at 37° C. The error bars represent the standard deviation of 4samples. Cylinders of 40% PEG were discontinued at 4 days due todisintegration.

FIGS. 24A, 24B and 24C are graphs which depict the change in dimensions,length (A), diameter (B) and wet weight (C) of 20% paclitaxel ladedPDLLA-PEG-PDLLA cylinders during the in vitro release of paclitaxel at37° C.

FIG. 25 is a graph which shows gel permeation chromatograms ofPDLLA-PEG-PDLLA cylinders (20% PEG, 1 mm diameter) loaded with 20%paclitaxel during the release in PBS albumin buffer at 37° C.

FIG. 26 is a table which shows the mass loss and polymer compositionchange of PDLLA-PEG-PDLLA cylinders (loaded with 20% paclitaxel) duringthe release into PBS albumin buffer at 37° C.

FIGS. 27A, 27B, 27C and 27D are SEMs of dried PDLLA-PEG-PDLLA cylinders(loaded with 20% paclitaxel, 1 mm in diameter) before and duringpaclitaxel release. A: 20% PEG, day 0; B: 30% PEG, day 0; C: 20% PEG,day 69; D: 30% PEG, day 69.

FIG. 28 is a graph which depicts the cumulative release of paclitaxelfrom 20% paclitaxel loaded PDLLA:PCL blends and PCL into PBS albuminbuffer at 37° C. The error bars represent the standard deviations of 4samples.

FIG. 29 is a table which shows the efficacy of paclitaxel loadedsurgicalpaste formulations applied locally tosubcutaneous tumor in mice.

FIG. 30A is a graph which depicts the time course of paclitaxel releasefrom 2.5 mg pellets of PCL. FIG. 30B is a graph which shows the percentpaclitaxel remaining in the pellet, over time.

FIG. 31A is a graph which shows the effect of MePEG on paclitaxelrelease from PCL paste leaded with 20% paclitaxel. FIG. 31B is a graphwhich shows the percent paclitaxel remaining in the pellet, over time.

FIGS. 32A and 32B are graphs which show the effect of variousconcentrations of MePEG in PCL in terms of melting point (32A) and timeto solidify (32B).

FIG. 33 is a graph which shows the effect of MePEG incorporation intoPCL on the tensile strength and time to fail of the polymer.

FIG. 34 is a graph which shows the effect of irradiation on paclitaxelrelease.

FIG. 35 is a graph which depicts the range of particle sizes for controlmicrospheres (PLLA:GA—85:15).

FIG. 36 is a graph which depicts the range of particle sizes for 20%paclitaxel loaded microspheres (PLLA:GA—85-15).

FIG. 37 is a graph which depicts the range of particle sizes for controlmicrospheres (PLLA:GA—85-15).

FIG. 38 is a graph which depicts the range of particle sizes for 20%paclitaxel loaded microspheres (PLLA:GA—85-15).

FIGS. 39A, 39B and 39C are graphs which depict the range of particlesizes for various ratios of PLLA and GA.

FIGS. 40A and 40B are graphs which depict the range of particle sizesfor various ratios of PLLA and GA

FIGS. 41A, 41B and 41C are graphs which depict the range of particlesizes for various ratios of PLLA and GA.

FIGS. 42A and 42B are graphs which depict the range of particle sizesfor various ratios of PLLA and GA

FIG. 43 is a table which shows the molecular weights, CMCs and maximumpaclitaxel loadings of selected diblock copolymers.

FIGS. 44A and 44B are graphs which depict the solubilization ofpaclitaxel crystals in water (37° C.) by the copolymers and CremophorEL. 44A; effect of the concentration of copolymer on Cremophor (20 hoursincubation); 44B: effect of time (copolymer or Cremophor concentration0.5%).

FIGS. 45A and 45B are graphs which depict the turbidity (uv-visabsorbance at 450 nm) of micellar paclitaxel solutions at roomtemperature (22° C.). Paclitaxel concentration was 2 mg/ml in water.Paclitaxel loading was 10% except MePEG 5000-30/70 where the loading was5%.

FIG. 46 is a graph which depicts paclitaxel release frompaclitaxel-nylon microcapsules.

FIG. 47 is a graph which plots the observed pseudo first order kineticdegradation of paclitaxel (20 μg ml⁻¹ in 10% HPβCD and 10% HPγCDsolutions at 37° C. and pH of 3.7 and 4.9, respectively.

FIG. 48 is a graph which shows the phase solubility for cyclodextrinsand paclitaxel in water at 37° C.

FIG. 49 is a graph which shows second order plots of the complexation ofpaclitaxel and γCD, HPβCD or HPγCD at 37° C.

FIG. 50 is a graph which shows the phase solubility for paclitaxel at37° C. and hydroxypropyl-α-cyclodextrin in 50:50 water:ethanolsolutions.

FIG. 51 is a graph which shows dissolution rate profiles of paclitaxelin 0, 5, 10 or 20% HPγCD solutions at 37° C.

FIGS. 51A and 51B are two photographs of a CAM having a tumor treatedwith control (unloaded) thermopaste. Briefly, in FIG. 51A the centralwhite mass is the tumor tissue. Note the abundance of blood vesselsentering the tumor from the CAM in all directions. The tumor induces theingrowth of the host vasculature through the production of “angiogenicfactors.” The tumor tissue expands distally along the blood vesselswhich supply it. FIG. 51B is an underside view of the CAM shown in 51A.Briefly, this view demonstrates the radial appearance of the bloodvessels which enter the tumor like the spokes of a wheel. Note that theblood vessel density is greater in the vicinity of the tumor than it isin the surrounding normal CAM tissue. FIGS. 51C and 51D are twophotographs of a CAM having a tumor treated with 20% paclitaxel-loadedthermopaste. Briefly, in FIG. 51C the central white mass is the tumortissue. Note the paucity of blood vessels in the vicinity of the tumortissue. The sustained release of the angiogenesis inhibitor is capableof overcoming the angiogenic stimulus produced by the tumor. The tumoritself is poorly vascularized and is progressively decreasing in size.FIG. 51D is taken from the underside of the CAM shown in 51C, anddemonstrates the disruption of blood flow into the tumor when comparedto control tumor tissue. Note that the blood vessel density is reducedin the vicinity of the tumor and is sparser than that of the normalsurrounding CAM tissue.

FIG. 52A is a graph which shows the effect of paclitaxel/PCL on tumorgrowth. FIGS. 52B and 52C are two photographs which show the effect ofcontrol, 10%, and 20% paclitaxel-loaded thermopaste on tumor growth.

FIG. 53 is a bar graph which depicts the size distribution ofmicrospheres by number (5% poly (ethylene-vinyl acetate) with 10 mgsodium suramin into 5% PVA).

FIG. 54 is a bar graph which depicts the size distribution ofmicrospheres by weight (5% poly (ethylene-vinyl acetate) with 10 mgsodium suramin into 5% PVA).

FIG. 55 is a graph which depicts the weight of encapsulation of SodiumSuramin in 50 mg poly (ethylene-vinyl acetate).

FIG. 56 is a graph which depicts the percent of encapsulation of SodiumSuramin in 50 mg poly (ethylene-vinyl acetate).

FIG. 57 is a bar graph which depicts the size distribution by weight of5% ELVAX microspheres containing 10 mg sodium suramin made in 5% PVAcontaining 10% NaCl.

FIG. 58 is a bar graph which depicts the size distribution by weight of5% microspheres containing 10 mg sodium suramin made in 5% PVAcontaining 10% NaCl.

FIG. 59 is a bar graph which depicts the size distribution by number of5% microspheres containing 10 mg sodium suramin made in 5% PVAcontaining 10% NaCl.

FIG. 60A is a photograph of Suramin and Cortisone Acetate on a CAM(Mag=8×). Briefly, this image shows an avascular zone treated with 20 μgof suramin and 70 μg of cortisone acetate in 0.5% methylcellulose. Notethe blood vessels located at the periphery of the avascular zone whichare being redirected away from the drug source. FIG. 60B is a photographwhich shows the vascular detail of the effected region at a highermagnification (Mag=20×). Note the avascular regions and the typical“elbowing” effect of the blood vessels bordering the avascular zone.

FIGS. 61A, B, C, D and E show the effect of MTX release from PCL overtime.

FIG. 62 is a photograph of 10% methotrexate-loaded microspheres madefrom PLA:GA (50:50); Inherent Viscosity “IV”=0.78.

FIG. 63 is a graph which depicts the release of 10% loaded vanadylsulfate from PCL.

FIG. 64 is a photograph of hyaluronic acid microspheres containingvanadium sulfate.

FIG. 65A is a graph which depicts the release of organic vanadate fromPCL. FIG. 65B depicts the percentage of organic vanadate remaining overa time course.

FIG. 66 is a photograph showing poly D,L, lactic acid microspherescontaining organic vanadate.

FIGS. 67A and 67B are graphs which show the time course of BMOV releasefrom PCL (150 mg slabs). (A) μg drug released or (B) % of drug remainingin slab. Initial loading of BMOV in PCL given by (◯), 5%; (●), 10%; (Δ),15%; (σ), 20%; ( ), 30% and (∇), 35%.

FIGS. 68A and 68B are graphs which show the time course of BMOV releasefrom 150 mg slabs of PCL:MEPEG (80:20, w:w) expressed as (A) μg drugreleased or (B) % drug remaining in slab. Initial loading of BMOV inPCL:MEPEG given by (◯), 5%; (●), 10%; (Δ), 15%; (σ), 20%.

FIGS. 69A, 69B and 69C are Scanning electron micrographs of (69A: top),BMOV crystals; (69B: middle) surface morphology of the PCL slabcontaining 20% BMOV at the start of the drug release experiment and(69C: bottom), surface morphology of the PCL slab containing 20% BMOV atthe end of the drug release experiment (72 days in PBS).

FIGS. 70A and 70B are two graphs which show the effect of increasingconcentration of BMOV on cell survival using 1 hour exposure of cells toBMOV (70A), or, continuous exposure to BMOV (70B). Cells described by(◯), HT-29 colon cells; (●), MCF-7 breast cells; (Δ), Skmes-1 non-smalllung cells and (σ), normal bone marrow cells.

FIG. 71 is a table which shows the effect of BMOV loaded paste on theweights of MDAY-D2 tumors grown in mice. Briefly, PCL paste (150 mg)containing either 25%, 30%, or 35% BMOV was injected subcutaneously intomice bearing MDAY-2 tumors. Tumor weights were determined after 10 daystreatment. This table shows the results from 2 separate experimentsusing (top table) 25% BMOV and (bottom table) 30% or 35% BMOV. Controldata describes mice treated with PCL containing no BMOV.

FIG. 72 is a table which sets forth the effects of BMOV loaded PCL:MePEGpaste on the weights of RIF-1 tumors grown in mice. Briefly, RIF-1tumors were grown in mice for 5 days at which time 90% of the tumor wassurgically removed and the resection site treated with 150 mg ofPCL:MePEG (80:20, w:w) paste containing either no BMOV (control) or 5%BMOV. Tumor regrowth was determined on days 4, 5 and 6 following thistreatment.

FIGS. 73A and 73B are two graphs. FIG. 73A shows the effect of increasedloading of BEMOV in PCL thermopaste (150 mg pellet) on the time courseof BEMOV released into 15 mL PBS/ALB. FIG. 73B also shows the effect ofincrease loading of BEMOV in PCL thermopaste (150 mg pellet) on the timecourse of BEMOV released into 15 mL PBS/ALB. Drug release is expressedas the % of BEMOV remaining in the pellet.

FIGS. 74A and 74B are two graphs. FIG. 74A shows the effect of increasedloading of V5 in PCL thermopaste (150 mg pellet) on the time course ofV5 released into 15 mL PBS/ALB. FIG. 74B also shows the effect ofincrease loading of V5 in PCL thermopaste (150 mg pellet) on the timecourse of V5 released into 15 mL PBS/ALB. Drug release is expressed asthe % of V5 remaining in the pellet.

FIGS. 75A and 75B are two graphs. FIG. 75A shows the effect of increasedloading of PRC-V in PCL thermopaste (150 mg pellet) on the time courseof PRC-V released into 15 mL PBS/ALB. FIG. 75B also shows the effect ofincrease loading of PRC-V in PCL thermopaste (150 mg pellet) on the timecourse of PRC-V released into 15 mL PBS/ALB. Drug release is expressedas the % of PRC-V remaining in the pellet.

FIGS. 76A, 76B, 76C and 76D are a series of graphs which show the effectof loading different concentrations of MePEG in PCL thermopaste (150 mgpellet) with 5% BMOV (76A), 10% BMOV (76B), 15% BMOV (76C), and 20% BMOV(76D) on the time course of BMOV released into 15 mL PBS/ALB.

FIGS. 77A, 77B, 77C and 77D are a series of graphs which show the effectof loading different concentrations of MePEG in PCL thermopaste (150 mgpellet) with 0% MePEG (77A), 5% MePEG (77B), 10% MePEG (77C), and 15%MePEG (77D) on the time course of BMOV released into 15 mL PBS/ALB. Drugrelease is expressed as the % of BMOV remaining in the pellet.

FIGS. 78A and 78B are photographs of fibronectin coated PLLAmicrospheres on bladder tissue (78A), and poly (L-lysine) microsphereson bladder tissue.

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention, it may be helpful to anunderstanding thereof to set forth definitions of certain terms thatwill be used hereinafter.

“Body passageway” as used herein refers to any of number of passageways,tubes, pipes, tracts, canals, sinuses or conduits which have an innerlumen and allow the flow of materials within the body. Representativeexamples of body passageways include arteries and veins, lacrimal ducts,the trachea, bronchi, bronchiole, nasal passages (including the sinuses)and other airways, eustachian tubes, the external auditory canal, oralcavities, the esophagus, the stomach, the duodenum, the small intestine,the large intestine, biliary tracts, the ureter, the bladder, theurethra, the fallopian tubes, uterus, vagina and other passageways ofthe female reproductive tract, the vasdeferens and other passageways ofthe male reproductive tract, and the ventricular system (cerebrospinalfluid) of the brain and the spinal cord.

“Therapeutic agent” as used herein refers to those agents which canmitigate, treat, cure, or prevent a given disease or condition.Representative examples of therapeutic agents are discussed in moredetail below, and include, for example, anti-angiogenic agents,anti-proliferative agents, anti-inflammatory agents, and antibiotics.

As noted above, the present invention provides methods for treating orpreventing diseases associated with body passageways, comprising thestep of delivering to an external portion of the body passageway (i.e.,a non-luminal surface), a composition comprising a therapeutic agent,and within preferred embodiments, a compositions comprising atherapeutic agent and a polymeric carrier. Briefly, delivery of atherapeutic agent to an external portion of a body passageway (e.g.,quadrantically or circumferentially) avoids many of the disadvantages oftraditional approaches which involve endoluminal manipulation. Inaddition, delivery of a therapeutic agent as described herein allows theadministration of greater quantities of the therapeutic agent with lessconstraint upon the volume to be delivered.

As discussed in more detail below, a wide variety of therapeutic agentsmay be delivered to external portions of body passageways, either withor without a carrier (e.g., polymeric), in order to treat or prevent adisease associated with the body passageway. Each of these aspects isdiscussed in more detail below.

Therapeutic Agents

As noted above, the present invention provides methods and compositionswhich utilize a wide variety of therapeutic agents. Within one aspect ofthe invention, the therapeutic agent is an anti-angiogenic factor.Briefly, within the context of the present invention anti-angiogenicfactors should be understood to include any protein, peptide, chemical,or other molecule which acts to inhibit vascular growth. A variety ofmethods may be readily utilized to determine the anti-angiogenicactivity of a given factor, including for example, chick chorioallantoicmembrane (“CAM”) assays. Briefly, a portion of the shell from a freshlyfertilized chicken egg is removed, and a methyl cellulose diskcontaining a sample of the anti-angiogenic factor to be tested is placedon the membrane. After several days (e.g., 48 hours), inhibition ofvascular growth by the sample to be tested may be readily determined byvisualization of the chick chorioallantoic membrane in the regionsurrounding the methyl cellulose disk. Inhibition of vascular growth mayalso be determined quantitatively, for example, by determining thenumber and size of blood vessels surrounding the methyl cellulose disk,as compared to a control methyl cellulose disk. Although anti-angiogenicfactors as described herein are considered to inhibit the formation ofnew blood vessels if they do so in merely a statistically significantmanner, as compared to a control, within preferred aspects suchanti-angiogenic factors completely inhibits the formation of new bloodvessels, as well as reduce the size and number of previously existingvessels.

In addition to the CAM assay described above, a variety of other assaysmay also be utilized to determine the efficacy of anti-angiogenicfactors in vivo, including for example, mouse models which have beendeveloped for this purpose (see Roberston et al., Cancer. Res.51:1339-1344, 1991).

A wide variety of anti-angiogenic factors may be readily utilized withinthe context of the present invention. Representative examples includeAnti-Invasive Factor, retinoic acid and derivatives thereof, Suramin,Tissue Inhibitor of Metalloproteinase-1, Tissue Inhibitor ofMetalloproteinase-2, Plasminogen Activator Inhibitor-1, PlasminogenActivator Inhibitor-2, compounds which disrupt microtubule function, andvarious forms of the lighter “d group” transition metals. These andother anti-angiogenic factors will be discussed in more detail below.

Briefly, Anti-Invasive Factor, or “AIF” which is prepared from extractsof cartilage, contains constituents which are responsible for inhibitingthe growth of new blood vessels. These constituents comprise a family of7 low molecular weight proteins (<50,000 daltons) (Kuettner and Pauli,“Inhibition of neovascularization by a cartilage factor” in Developmentof the Vascular System, Pitman Books (CIBA Foundation Symposium 100),pp. 163-173, 1983), including a variety of proteins which haveinhibitory effects against a variety of proteases (Eisentein et al, Am.J. Pathol. 81:337-346, 1975; Langer et al., Science 193:70-72, 1976; andHorton et al., Science 199:1342-1345, 1978). AIF suitable for use withinthe present invention may be readily prepared utilizing techniques knownin the art (e.g., Eisentein et al, supra; Kuettner and Pauli, supra; andLanger et al., supra). Purified constituents of AIF such asCartilage-Derived Inhibitor (“CDI”) (see Moses et al., Science248:1408-1410, 1990) may also be readily prepared and utilized withinthe context of the present invention.

Retinoic acids alter the metabolism of extracellular matrix components,resulting in the inhibition of angiogenesis. Addition of prolineanalogs, angiostatic steroids, or heparin may be utilized in order tosynergistically increase the anti-angiogenic effect of transretinoicacid. Retinoic acid, as well as derivatives thereof which may also beutilized in the context of the present invention, may be readilyobtained from commercial sources, including for example, Sigma ChemicalCo. (# R2625).

Suramin is a polysulfonated naphthylurea compound that is typically usedas a trypanocidal agent. Briefly, Suramin blocks the specific cellsurface binding of various growth factors such as platelet derivedgrowth factor (“PDGF”), epidermal growth factor (“EGF”), transforminggrowth factor (“TGF-β”), insulin-like growth factor (“IGF-1”), andfibroblast growth factor (“βFGF”). Suramin may be prepared in accordancewith known techniques, or readily obtained from a variety of commercialsources, including for example Mobay Chemical Co., New York. (seeGagliardi et al., Cancer Res. 52:5073-5075, 1992; and Coffey, Jr., etal., J. of Cell. Phys. 132:143-148, 1987).

Tissue Inhibitor of Metalloproteinases-1 (“TIMP”) is secreted byendothelial cells which also secrete MMPases. TIMP is glycosylated andhas a molecular weight of 28.5 kDa. TIMP-1 regulates angiogenesis bybinding to activated metalloproteinases, thereby suppressing theinvasion of blood vessels into the extracellular matrix. TissueInhibitor of Metalloproteinases-2 (“TIMP-2”) may also be utilized toinhibit angiogenesis. Briefly, TIMP-2 is a 21 kDa nonglycosylatedprotein which binds to metalloproteinases in both the active and latent,proenzyme forms. Both TIMP-1 and TIMP-2 may be obtained from commercialsources such as Synergen, Boulder, Colo.

Plasminogen Activator Inhibitor-1 (PA) is a 50 kDa glycoprotein which ispresent in blood platelets, and can also be synthesized by endothelialcells and muscle cells. PAI-1 inhibits t-PA and urokinase plasminogenactivator at the basolateral site of the endothelium, and additionallyregulates the fibrinolysis process. Plasminogen Activator Inhibitor-2(PAI-2) is generally found only in the blood under certain circumstancessuch as in pregnancy, and in the presence of tumors. Briefly, PAI-2 is a56 kDa protein which is secreted by monocytes and macrophages. It isbelieved to regulate fibrinolytic activity, and in particular inhibitsurokinase plasminogen activator and tissue plasminogen activator,thereby preventing fibrinolysis.

Therapeutic agents of the present invention also include compounds whichdisrupt microtubule function. Representative examples of such compoundsinclude estramustine (available from Sigma; Wang and Stearns Cancer Res.48:6262-6271, 1988), epothilone, curacin-A, colchicine, methotrexate,and paclitaxel, vinblastine, vincristine, D₂O and4-tert-butyl-[3-(2-chloroethyl) ureido] benzene (“tBCEU”). Briefly, suchcompounds can act in several different manners. For example, compoundssuch as colchicine and vinblastine act by depolymerizing microtubules.

Within one preferred embodiment of the invention, the therapeutic agentis paclitaxel, a compound which disrupts microtubule formation bybinding to tubulin to form abnormal mitotic spindles. Briefly,paclitaxel is a highly derivatized diterpenoid (Wani et al., J. Am.Chem. Soc. 93:2325, 1971) which has been obtained from the harvested anddried bark of Taxus brevifolia (Pacific Yew.) and Taxomyces Andreanaeand Endophytic Fungus of the Pacific Yew (Stierle et al., Science60:214-216, 1993). “Paclitaxel” (which should be understood herein toinclude prodrugs, analogues and derivatives such as, for example,TAXOL®, TAXOTERE®, 10-desacetyl analogues of paclitaxel and3′N-desbenzoyl-3′N-t-butoxy carbonyl analogues of paclitaxel) may bereadily prepared utilizing techniques known to those skilled in the art(see WO 94/07882, WO 94/07881, WO 94/07880, WO 94/07876, WO 93/23555, WO93/10076, WO94/00156, WO 93/24476, EP 590267, WO 94/20089; U.S. Pat.Nos. 5,294,637, 5,283,253, 5,279,949, 5,274,137, 5,202,448, 5,200,534,5,229,529, 5,254,580, 5,412,092, 5,395,850, 5,380,751, 5,350,866,4,857,653, 5,272,171, 5,411,984, 5,248,796, 5,248,796, 5,422,364,5,300,638, 5,294,637, 5,362,831, 5,440,056, 4,814,470, 5,278,324,5,352,805, 5,411,984, 5,059,699, 4,942,184; Tetrahedron Letters35(52):9709-9712, 1994; J. Med. Chem. 35:4230-4237, 1992; J. Med. Chem.34:992-998, 1991; J. Natural Prod. 57(10):1404-1410, 1994; J. NaturalProd. 57(11):1580-1583, 1994; J. Am. Chem. Soc. 110:6558-6560, 1988), orobtained from a variety of commercial sources, including for example,Sigma Chemical Co., St. Louis, Mo. (T7402—from Taxus brevifolia).

Representative examples of such paclitaxel derivatives or analoguesinclude 7-deoxy-docetaxol, 7,8-Cyclopropataxanes, N-Substituted2-Azetidones, 6,7-Epoxy Paclitaxels, 6,7-Modified Paclitaxels,10-Desacetoxytaxol, 10-Deacetyltaxol (from 10-deacetylbaccatin III),Phosphonooxy and Carbonate Derivatives of Taxol, Taxol 2′,7-di(sodium1,2-benzenedicarboxylate,10-desacetoxy-11,12-dihydrotaxol-10,12(18)-diene derivatives,10-desacetoxytaxol, Protaxol (2′-and/or 7-O-ester derivatives),(2′-and/or 7-O-carbonate derivatives), Asymmetric Synthesis of TaxolSide Chain, Fluoro Taxols, 9-deoxotaxane, (13-acetyl-9-deoxobaccatineIII, 9-deoxotaxol, 7-deoxy-9-deoxotaxol,10-desacetoxy-7-deoxy-9-deoxotaxol, Derivatives containing hydrogen oracetyl group and a hydroxy and tert-butoxycarbonylamino, sulfonated2′-acryloyltaxol and sulfonated 2′-O-acyl acid taxol derivatives,succinyltaxol, 2′-γ-aminobutyryltaxol formate, 2′-acetyl taxol, 7-acetyltaxol, 7-glycine carbamate taxol, 2′-OH-7-PEG(5000) carbamate taxol,2′-benzoyl and 2′,7-dibenzoyl taxol derivatives, other prodrugs(2′-acetyltaxol; 2′,7-diacetyltaxol; 2′ succinyltaxol;2′-(beta-alanyl)-taxol); 2′ gamma-aminobutyryltaxol formate; ethyleneglycol derivatives of 2′-succinyltaxol; 2′-glutaryltaxol;2′-(N,N-dimethylglycyl) taxol; 2′-[2-(N,N-dimethylamino)propionyl]taxol;2′ orthocarboxybenzoyl taxol; 2′ aliphatic carboxylic acid derivativesof taxol, Prodrugs {2′ (N,N-diethylaminopropionyl)taxol, 2′(N,N-dimethylglycyl)taxol, 7(N,N-dimethylglycyl)taxol,2′,7-di-(N,N-dimethylglycyl)taxol, 7(N,N-diethylaminopropionyl)taxol,2′,7-di(N,N-diethylaminopropionyl)taxol, 2′-(L-glycyl)taxol,7-(L-glycyl)taxol, 2′,7-di(L-glycyl)taxol, 2′-(L-alanyl)taxol,7-(L-alanyl)taxol, 2′,7-di(L-alanyl)taxol, 2′-(L-leucyl)taxol,7-(L-leucyl)taxol, 2′,7-di(L-leucyl)taxol, 2′-(L-isoleucyl)taxol,7-(L-isoleucyl)taxol, 2′,7-di(L-isoleucyl)taxol, 2′-(L-valyl)taxol,7-(L-valyl)taxol, 2′7-di(L-valyl)taxol, 2′-(L-phenylalanyl)taxol,7-(L-phenylalanyl)taxol, 2′,7-di(L-phenylalanyl)taxol,2′-(L-prolyl)taxol, 7-(L-prolyl)taxol, 2′,7-di(L-prolyl)taxol,2′-(L-lysyl)taxol, 7-(L-lysyl)taxol, 2′,7-di(L-lysyl)taxol,2′-(L-glutamyl)taxol, 7-(L-glutamyl)taxol, 2′,7-di(L-glutamyl)taxol,2′-(L-arginyl)taxol, 7-(L-arginyl)taxol, 2′,7-di(L-arginyl)taxol}, Taxolanalogs with modified phenylisoserine side chains, taxotere,(N-debenzoyl-N-tert-(butoxycaronyl)-10-deacetyltaxol, and taxanes (e.g.,baccatin III, cephalomannine, 10-deacetylbaccatin III, brevifoliol,yunantaxusin and taxusin)

Other therapeutic agents which may be utilized within the presentinvention include lighter “d group” transition metals, such as, forexample, vanadium, molybdenum, tungsten, titanium, niobium, and tantalumspecies. Such transition metal species may form transition metalcomplexes. Suitable complexes of the above-mentioned transition metalspecies include oxo transition metal complexes.

Representative examples of vanadium complexes include oxo vanadiumcomplexes such as vanadate and vanadyl complexes. Suitable vanadatecomplexes include metavanadate (i.e., VO₃ ⁻) and orthovanadate (i.e.,VO₄ ³⁻) complexes such as, for example, ammonium metavanadate (i.e.,NH₄VO₃), sodium metavanadate (i.e., NaVO₃), and sodium orthovanadate(i.e., Na₃VO₄). Suitable vanadyl (i.e., VO²⁺) complexes include, forexample, vanadyl acetylacetonate and vanadyl sulfate including vanadylsulfate hydrates such as vanadyl sulfate mono- and trihydrates,Bis[maltolato(oxovanadium)] (IV)] (“BMOV”),Bis[(ethylmaltolato)oxovanadium] (IV) (“BEOV”), and Bis(cysteine, amideN-octyl)oxovanadium (IV) (“naglivan”).

Representative examples of tungsten and molybdenum complexes alsoinclude oxo complexes. Suitable oxo tungsten complexes include tungstateand tungsten oxide complexes. Suitable tungstate (i.e., WO₄ ²⁻)complexes include ammonium tungstate (i.e., (NH₄)₂WO₄), calciumtungstate (i.e., CaWO₄), sodium tungstate dihydrate (i.e., Na₂WO₄.2H₂O),and tungstic acid (i.e., H₂WO₄). Suitable tungsten oxides includetungsten (IV) oxide (i.e., WO₂) and tungsten (VI) oxide (i.e., WO₃).Suitable oxo molybdenum complexes include molybdate, molybdenum oxide,and molybdenyl complexes. Suitable molybdate (i.e., MoO₄ ²⁻) complexesinclude ammonium molybdate (i.e., (NH₄)₂MoO₄) and its hydrates, sodiummolybdate (i.e., Na₂MoO₄) and its hydrates, and potassium molybdate(i.e., K₂MoO₄) and its hydrates. Suitable molybdenum oxides includemolybdenum (VI) oxide (i.e., MoO₂), molybdenum (VI) oxide (i.e., MoO₃),and molybdic acid. Suitable molybdenyl (i.e., MoO₂ ²⁺) complexesinclude, for example, molybdenyl acetylacetonate. Other suitabletungsten and molybdenum complexes include hydroxo derivatives derivedfrom, for example, glycerol, tartaric acid, and sugars.

A wide variety of other anti-angiogenic factors may also be utilizedwithin the context of the present invention. Representative examplesinclude Platelet Factor 4 (Sigma Chemical Co., #F1385); ProtamineSulphate (Clupeine) (Sigma Chemical Co., #P4505); Sulphated ChitinDerivatives (prepared from queen crab shells), (Sigma Chemical Co.,#C3641; Murata et al., Cancer Res. 51:22-26, 1991); SulphatedPolysaccharide Peptidoglycan Complex (SP-PG) (the function of thiscompound may be enhanced by the presence of steroids such as estrogen,and tamoxifen citrate); Staurosporine (Sigma Chemical Co., #S4400);Modulators of Matrix Metabolism, including for example, proline analogs{[(L-azetidine-2-carboxylic acid (LACA) (Sigma Chemical Co., #A0760)),cishydroxyproline, d,L-3,4-dehydroproline (Sigma Chemical Co., #D0265),Thiaproline (Sigma Chemical Co., #T0631)], α,α-dipyridyl (Sigma ChemicalCo., #D7505), β-aminopropionitrile fumarate (Sigma Chemical Co.,#A3134)]}; MDL 27032 (4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; MerionMerrel Dow Research Institute); Methotrexate (Sigma Chemical Co.,#A6770; Hirata et al., Arthritis and Rheumatism 32:1065-1073, 1989);Mitoxantrone (Polyerini and Novak, Biochem. Biophys. Res. Comm.140:901-907); Heparin (Folkman, Bio. Phar. 34:905-909, 1985; SigmaChemical Co., #P8754); Interferons (e.g., Sigma Chemical Co., #13265); 2Macroglobulin-serum (Sigma Chemical Co., #M7151); ChIMP-3 (Pavloff etal., J. Bio. Chem. 267:17321-17326, 1992); Chymostatin (Sigma ChemicalCo., #C7268; Tomkinson et al., Biochem J. 286:475-480, 1992);β-Cyclodextrin Tetradecasulfate (Sigma Chemical Co., #C4767);Eponemycin; Camptothecin; Fumagillin and derivatives (Sigma ChemicalCo., #F6771; Canadian Patent No. 2,024,306; Ingber et al., Nature348:555-557, 1990); Gold Sodium Thiomalate (“GST”; Sigma:G4022;Matsubara and Ziff, J. Clin. Invest. 79:1440-1446, 1987);(D-Penicillamine (“CDPT”; Sigma Chemical Co., #P4875 or P5000(HCl));β-1-anticollagenase-serum; α2-antiplasmin (Sigma Chem. Co.: A0914;Holmes et al., J. Biol. Chem. 262(4):1659-1664, 1987); Bisantrene(National Cancer Institute); Lobenzarit disodium(N-(2)-carboxyphenyl-4-chloroanthronilic acid disodium or “CCA”;Takeuchi et al., Agents Actions 36:312-316, 1992); Thalidomide;Angostatic steroid; AGM-1470; carboxynaminolmidazole; andmetalloproteinase inhibitors such as BB94, estrogen and estrogenanalogues, antiestrogens, antioxidants, bioflavonoids (Pycnogenol),ether lipids (s-phosphonate, ET-18-OCH₃), tyrosine kinase inhibitors(genisteine, erbstatin, herbamycin A, lavendustine-c,hydroxycinnamates), a chemokines [Human interferon-inducible protein 10(IP-10)], —C—X—C—Chemokines (Gro-beta), Nitric Oxide, Antifungal Agents(Radicicol), 15-deoxyspergualin, Metal Complexes (Titanocenedichloride-cyclopentadienyl titanium dichloride), TriphenylmethaneDerivatives (aurintricarboxylic acid), Linomide, Thalidomide, IL-12,Heparinase, Angiostatin, Antimicrobial Agents (Minocycline), PlasmaProteins (Apolipoprotein E), Anthracyclines (TAN-1120),Proliferin-Related Protein, FR-111142, Saponin of Panax ginseng(Ginsenoside-Rb2), Pentosan polysulfate

Compositions of the present invention may also contain a wide variety ofother therapeutic agents, including for example: α-adrenergic blockingagents, angiotensin II receptor antagonists and receptor antagonists forhistamine, serotonin, endothelin; inhibitors of the sodium/hydrogenantiporter (e.g., amiloride and its derivatives); agents that modulateintracellular Ca²⁺ transport such as L-type (e.g., diltiazem,nifedipine, verapamil) or T-type Ca²⁺ channel blockers (e.g.,amiloride), calmodulin antagonists (e.g., H₇) and inhibitors of thesodium/calcium antiporter (e.g., amiloride); ap-1 inhibitors (fortyrosine kinases, protein kinase C, myosin light chain kinase,Ca²⁺/calmodulin kinase II, casein kinase II); anti-depressants (e.g.,amytriptyline, fluoxetine, LUVOX® and PAXIL®); cytokine and/or growthfactors, as well as their respective receptors, (e.g., the interleukins,α, β or γ-IFN, GM-CSF, G-CSF, epidermal growth factor, transforminggrowth factors alpha and beta, TNF, and antagonists of vascularepithelial growth factor, endothelial growth factor, acidic or basicfibroblast growth factors, and platelet dervived growth factor);inhibitors of the IP₃ receptor (e.g., heparin); protease and collagenaseinhibitors (e.g., TIMPs, discussed above); nitrovasodilators (e.g.,isosorbide dinitrate); anti-mitotic agents (e.g., colchicine,anthracyclines and other antibiotics, folate antagonists and otheranti-metabolites, vinca alkaloids, nitrosoureas, DNA alkylating agents,topoisomerase inhibitors, purine antagonists and analogs, pyrimidineantagonists and analogs, alkyl sulfonates); immunosuppressive agents(e.g., adrenocorticosteroids, cyclosporine); sense or antisenseoligonucleotides (e.g., DNA, RNA, nucleic acid analogues (e.g., peptidenucleic acids) or any combinations of these); and inhibitors oftranscription factor activity (e.g., lighter d group transition metals).

Other therapeutic agents that can be utilized within the presentinvention include a wide variety of antibiotics, includingantibacterial, antimicrobial, antiviral, antiprotozoal and antifungalagents. Representative examples of such agents include systemicantibiotics such as aminoglycosides (e.g., streptomycin, amikacin,gentamicin, netilmicin, tobramycin); 1st, 2nd, and 3rd generationcephalosporins (e.g., cephalothin, cefazolin, cephapirin, cephradine,cephalexin, cefadroxil, cefaclor, cefamandole, cefuroxime, cefuroximeaxetil, cefonicid, ceforanide, cefoxitin, cefotaxime, cefotetan,ceflizoxime, cefoperazone, ceftazidime, ceftriaxone, moxalactam, othersemisynthetic cephalosporins such as cefixime and cefpodoxime proxetil);penicillins (e.g., penicillin G (benzathine and procaine salts),cloxacillin, dicloxacillin, methicillin, nafcillin, oxacillin,penicillin V, ampicillin, amoxicillin, bacampicillin, cyclacillin,carbenicillin, ticarcillin, mezlocillin, piperacillin, azlocillin,amdinocillin, and penicillins combined with clavulanic acid); quinolones(e.g., cinoxacin, ciprofloxacin, nalidixic acid, norfloxacin, pipemidicacid, perloxacin, fleroxacin, enoxacin, ofloxacin, tosufloxacin,lomefloxacin, stereoisomers of the quinolones); sulfonamides (e.g.,sulfacytine, sulfamethizole, sulfamethoxazole, sufisoxazole,sulfasalazine, and trimethoprim plus sulfamethoxazole combinations);tetracyclines (e.g., doxycycline, demeclocycline, methacycline,minocycline, oxytetracycline, tetracycline); macrolides (e.g.,erythromycins, other semisythetic macrolides such as azithromycin andclarithromycin); monobactams (new synthetic class) (e.g., aztreonam,loracarbef); and miscellaneous agents such as actinomycin D,doxorubicin, mitomycin C, novobiocin, plicamycin, rifampin, bleomycin,chloramphenicol, clindamycin, oleandomycin, kanamycin, lincomycin,neomycin, paromomycin, spectinomycin, troleandomycin, amphotericin B,colistin, nystatin, polymyxin B, griseofulvin, aztreonam, cycloserine,clindamycin, colistimethate, imipenem-cilastatin, methenamine,metronidazole, nitrofurantoin, rifabutan, spectinomycin, trimethoprim,bacitracin, vancomycin, other β-lactam antibiotics.

Further therapeutic agents that can be utilized within the presentinvention include topical antibiotics such as bacitracin, zinc,neomycin, mupirocin, clindamcin; antipathogenic polypeptides such ascecropionins, mangainins; and Antitubercular agents such assulfadimethoxine, sulfisoxazole, sulfisomidine, ethambutorhydrochloride, isoniazide, calcium paraminosalicylate.

Other therapeutic agents that can be utilized within the presentinvention include antibiotics such as iodine, povidone iodine, boricacid, sodium borate, oxydale, potassium permanganate, ethanol,isopropanol, formalin, cresol, dimazole, siccanin,phenyliodoundecynoate, hexachlorophene, resorcin, benzethonin chloride,sodium lauryl sulfate, mercuric chloride, mercurochrome, silversulfadiazine and other inorganic and organic silver and zinc salts,salts of mono- and divalent cations, chlorhexidine gluconate,alkylpolyaminoethylglycine hydrochloride, benzalkonium chloride,nitrofurazone, nystatin, acesulfamin, clotrimazole, sulfamethizole,sulfacetamide, diolamine, tolnaftate, pyrrolnitrin, undecylenic acid,microazole, variotin, haloprogin, and dimazole, (meclocycline,trichomycin and pentamycin), penicillins. Antifungal agents includeflucytosine, fluconazole, griseofluvin, ketoconazole and miconazole.Antiviral and AIDS agents include acyclovir, amantadine, didanosine(formerly ddI), griseofulvin, flucytosine, foscamet, ganciclovir,idoxuridine, miconazole, clotrimazole, pyrimethamine, ribavirin,rimantadine, stavudine (formerly d4T), trifluridine, trisulfapyrimidine,valacyclovir, vidarabine, zalcitabine (formerly ddC) and zidovudine(formerly AZT). Adjunct therapeutic agents for AIDS (e.g.,erythropoietin; fluconazole (antifungal); interferon alpha-2a and -2b(Kaposi's sarcoma); atovaquone, pentamidine and trimetrexate(antiprotozoal); megestraol acetate (appetite enhancer); rifabutin(antimycobacterial). Representative examples of antiprotozoal agentsinclude: pentamidine isethionate, quinine, chloroquine, and mefloquine.

Other therapeutic agents that can be utilized within the presentinvention include anti-proliferative, anti-neoplastic orchemotherapeutic agents. Representative examples of such agents includeandrogen inhibitors, antiestrogens and hormones such as flutamide,leuprolide, tamoxifen, estradiol, estramustine, megestrol,diethylstilbestrol, testolactone, goserelin, medroxyprogesterone;Cytotoxic agents such as altretamine, bleomycin, busulfan, carboplatin,carmustine(BiCNU), cisplantin, cladribine, dacarbazine, dactinomycin,daunorubicin, doxorubicin, estramustine, etoposide, lomustine,cyclophosphamide, cytarabine, hydroxyurea, idarubicin, interferonalpha-2a and -2b, ifosfamide, mitoxantrone, mitomycin, paclitaxel,streptozocin, teniposide, thiotepa, vinblastine, vincristine,vinorelbine; Antimetabolites and antimitotic agents such as floxuridine,5-fluorouracil, fluarabine, interferon alpha-2a and -2b, leucovorin,mercaptopurine, methotrexate, mitotane, plicamycin, thioguanine,colchicine, anthracyclines and other antibiotics, folate antagonists andother anti-metabolites, vinca alkaloids, nitrosoureas, DNA alkylatingagents, purine antagonists and analogs, pyrimidine antagonists andanalogs, alkyl solfonates; enzymes such as asparaginase, pegaspargase;radioactive agents (e.g., Cu-64, Ga-67, Ga-68, Zr-89, Ru-97, Tc-99m,Rh-105, Pd-109, In-111, I-123, I-125, I-131, Re-186, Re-188, Au-198,Au-199, Pb-203, At-211, Pb-212 and Bi-212), toxins (e.g., ricin, abrin,diphtheria toxin, cholera toxin, gelonin, pokeweed antiviral protein,tritin, Shigella toxin, and Pseudomonas exotoxin A), adjunct therapeuticagents such as granisetron and ondansetron (antinauseants, antiemetics),dexrazoxane (cardiomyopathy), gallium nitrate (hypercalcemia), GCSF andGMSCF (chemotherapy and BMT), IL-1 alpha, IL-2, IL-3, IL-4, levamisole,pilocarpine (saliva generation in radiation therapy setting), strontium89 (bone tumors).

Further therapeutic agents that can be utilized within the presentinvention include Cardiovascular agents; Antihypertensive agents;Adrenergic blockers and stimulators (e.g., doxazosin, guanadrel,guanethidine, pheoxybenzamine, prazosin plus polythiazide, terazosin,methyldopa, clonidine, guanabenz, guanfacine); Alpha-/beta-adrenergicblockers (e.g., Labetalol); angiotensin converting enzyme (ACE)inhibitors (e.g., benazepril, catopril, enalapril, enalaprilat,fosinopril, lisinopril, moexipril, quinapril, ramipril, and combinationswith calcium channel blockers and diuretics; ACE-receptor antagonists(e.g., losartan); Beta blockers (e.g., acebutolol, atenolol, betaxolol,bisoprolol, carteolol, esmolol, fimolol, pindolol, propranolol,penbatolol, metoprolol, nadolol, sotalol); Calcium channel blockers(e.g., Amiloride, amlodipine, bepridil, diltiazem, isradipine,nifedipine, verapamil, felodipine, nicardipine, nimodipine);Antiarrythmics, groups I-IV (e.g., bretylium, disopyramide, encainide,flecainide, lidocaine, mexiletine, moricizine, propafenone,procainamide, quinidine, tocainide, esmolol, propranolol, acebutolol,amiodarone, sotalol, verapamil, diltiazem, pindolol, bupranololhydrochloride, trichlormethiazide, furosemide, prazosin hydrochloride,metoprolol tartrate, carteolol hydrochloride, oxprenolol hydrochloride,and propranolol hydrochloride); and miscellaneous antiarrythmics andcardiotonics (e.g., adenosine, digoxin; metildigoxin, caffeine, dopaminehydrochloride, dobutamine hydrochloride, octopamine hydrochloride,diprophylline, ubidecarenon, digitalis).

Other therapeutic agents that can be utilized within the presentinvention include diuretics (e.g., acetazolamide, amiloride, triamtereneplus hydrochlorothiazide combinations, spironolactone plushydrochlorothiazide combinations, torsemide, furosemide, ethacrynate,bumetanide, triamterene, methylchorothizide, hydrochlorothiazide,metdazone, chlorthalidone, hydroflumethiazide, metolazone,methyclothiazide, polythiazide, quinithazone, trichlormethiazide,benroflumethiazide, benzthiazide); hypotensive diuretics (e.g.,mefruside, penflutizide, bumetamide, hydrothiazide, bentroflumethiazide,reserpine); Inotropic agents (e.g., digoxin, digitoxin, dobutamine,amrinone, milrinone); vasodilators (e.g., papaverine, isosorbide mono-and dinitrates, nitroglycerin, dizoxide, hydralazine, minoxidil,nitroprusside, prazosin, terazosin, 1,2,3-propanetriolmononitrate,1,2,3-propanetrioInitrate and their ester derivatives, pentaerythritoltetranitrate, hepronicate, molsidomine, nicomol, simfibrate, diltiazemhydrochloride, cinnarizine, dipyridamole, trapidil, trimetazidinehydrochloride, carbocromene, prenylamine lactate, dilazepdihydrochloride); vasopressors (e.g., metaraminol, isoproterenol,phenylephrine, methaxamine); anticoagulant and thrombolytic agents(e.g., tissue plasminogen activator(TPA), urokinase, streptokinase,pro-urokinase, urokinase, heparin, warfarin); Calmodulin antagonists(e.g., H₇); inhibitors of the sodium/calcium antiporter (e.g.,Amiloride); and inhibitors of the ryanodine receptor (e.g., Ryanodine);inhibitors of the IP₃ receptor (e.g., Heparin).

Other therapeutic agents that can be utilized within the presentinvention include anti-inflammatory agents. Representative examples ofsuch agents include nonsteroidal agents (“NSAIDS”) such as salicylates(e.g., salsalate, mesalamine, diflunisal, choline magnesiumtrisalicylate), diclofenac, diflunisal, etodolac, fenoprofen,flurbiprofen, ibuprofen, indomethacin, mefenamic acid, nabumetone,naproxen, piroxicam, phenylbutazone, ketoprofen, S-ketoprofen, ketorolactromethamine, sulindac, tolmetin). Other anti-inflammatory drugs includesteroidal agents such as beclomethasone, betamethasone, cortisone,dexamethasone, fluocinolone, flunisolide, fluticasone proprionate,fluorinated-corticoids, triamcinolone-diacetate, hydorcortisone,prednisolone, methylprednisolone and prednisone. Immunosuppressiveagents (e.g., adenocorticosteroids, cyclosporin); and antihistamines anddecongestants (e.g., astemizole(histamine H1-receptor antagonist),azatidine, brompheniramine, clemastine, chlorpheniramine, cromolyn,cyproheptadine, diphenylimidazole, diphenhydramine hydrochloride,hydroxyzine, glycyrrhetic acid, homochlorocyclizine hydrochloride,ketotifen, loratadine, naphazoline, phenindamine, pheniramine,promethazine, terfenadine, trimeprazine, tripelennamine, tranilast, andthe decongestants phenylpropanolamine and pseudo ephedrine.

Further therapeutic agents that can be utilized within the presentinvention include central nervous system agents. Representative examplesof such agents include anti-depressants (e.g., Prozac, Paxil, Luvox,Mannerex and Effexor); CNS stimulants (e.g., pemoline, methamphetamine,dextroamphetamine); hypnotic agents (e.g., pentobarbital, estazolam,ethchlorynol, flurazepam, propofol, secobarbital, temazepam, triazolam,quazepam, zolpidem tartrate); antimanic agents (e.g., lithium);sedatives and anticonvulsant barbiturates (e.g., pentobarbitol,phenobarbital, secobarbital, mephobarbital, butabarbital primidone,amobarbital); non-barbiturate sedatives (e.g., diphehydramine,doxylamine, midazolam, diazepam, promethazine, lorazepam, temazepam);and other miscellaneous hypnotics and sedatives (e.g., methaqualone,glutethimide, flurazepam, bromovalerylurea, flurazepam, hydrochloride,haloxazolam, triazolam, phenobarbital, chloral hydrate, nimetazepam,estazolam).

Other therapeutic agents that can be utilized within the presentinvention include Alzheimer's agents such as tacrine (reversiblecholinesterase inhibitor); Parkinson's disease agents such asamantadine, bromocriptine mesylate, biperiden, benztropine mesylate,carbidopa-levodopa, diphenhydramine, hyoscyamine, levodopa, pergolidemesylate, procyclidine, selegiline HCl, trihexyphenidyl HCl; and othermiscellaneous CNS agents such as fluphenazine, flutazolam,phenobarbital, methylphenobarbital, thioridazine, diazepam,benzbromarone, clocapramine hydrochloride, clotiazepam, chlorpromazine,haloperidol, lithium carbonate.

Further therapeutic agents that can be utilized within the presentinvention include anti-migraine agents (e.g., ergotamine, methylsergide,propranolol, dihydroergotamine, Sertroline and Immitrex); Post-cerebralembolism agents (e.g., nicardipine hydrochloride, cinepazide maleate,pentoxifylline, ifenprodil tartrate); local anesthetics (e.g.,lidocaine, benzocaine, ethyl aminobenzoate, procaine hydrochloride,dibucaine, procaine; antiulcer/antireflux agents (e.g., Losec(Omeprazole), aceglutamide aluminum, cetraxate hydrochloride,pirenzepine hydrochloride, cimetidine, famotidine, metoclopramide,ranitidine, L-glutamine, gefamate, and any stereoisomer of thesecompounds, and the pharmaceutically acceptable salts of these compounds,such compound used singly or in combination of more than one compound,properly chosen); protease inhibitors (e.g., serine protease,metalloendoproteases and aspartyl proteases (such as HIV protease, reninand cathepsin) and thiol protease inhibitors (e.g.,benzyloxycarbonyl-leu-norleucinal (calpeptin) andacetyl-leu-leu-norleucinal); phosphodiesterase inhibitors (e.g.,isobutyl methylxanthine); Phenothiazines; growth factor receptorantagonists (e.g., platelet-derived growth factor (PDGF), epidermalgrowth factor, interleukins, transforming growth factors alpha and beta,and acidic or basic fibroblast growth factors); antisenseoligonucleotides (e.g., sequences complementary to portions of mRNAencoding DPGF or other growth factors); and protein kinase inhibitors(e.g., For tyrosine kinases, protein kinase C, myosin light chainkinase, Ca²⁺/calmodulin kinase II, casein kinase II);

Other therapeutic agents that can be utilized within the presentinvention include anti-tissue damage agents. Representative examples ofsuch agents include Superoxide dismutase; Immune Modulators (e.g.,lymphokines, monokines, interferon α, β, τ-1b, α-n3, α-2b, α-2b; GrowthRegulators (e.g., IL-2, tumor necrosis factor, epithelial growth factor,somatrem, fibronectin, GM-CSF, CSF, platelet derived growth factor,somatotropin, rG-CSF, epidermal growth factor, IGF-1).

Other therapeutic agents that can be utilized within the presentinvention include monoclonal and polyclonal antibodies (e.g., thoseactive against: venoms, toxins, tumor necrosis factor, bacteria);hormones (e.g., estrogen, progestin, testosterone, human growth hormone,epinephrine, levarterenol, thyroxine, thyroglobulin, oxytocin,vasopressin, ACTH, somatropin, thyrotropin, insulin, parathyrin,calcitonin); vitamins (e.g., vitamins A, B and its subvitamins, C, D, E,F, G, J, K, N, P, PP, T, U and their subspecies); amino acids such asarginine, histidine, proline, lysine, methionine, alanine,phenylalanine, aspartic acid, glutamic acid, glutamine, threonine,tryptophan, glycine, isoleucine, leucine, valine; Prostaglandins (e.g.,E₁, E₂, F_(2α), I₂); enzymes such as pepsin, pancreatin, rennin, papain,trypsin, pancrelipase, chymopapain, bromelain, chymotrypsin,streptokinase, urokinase, tissue plasminogen activator, fibrinolysin,desoxyribonuclease, sutilains, collagenase, asparaginase, heparinase;buffers and salts (e.g., NaCl, cations including: Na⁺, K⁺, Ca⁺⁺, Mg⁺⁺,Zn⁺⁺, NH₄ ⁺ triethanolamine, anions including: phosphate, sulfate,chloride, citrate, ascorbate, acetate, borate, carbonate ions);preservatives (e.g., benzalkonium chloride, Na or K bisulfite, Na or Kthiosulfate, parabans); antigout agents (e.g., allopurinol, cochicine,probenicid, sulfinpyrazone); antidepressant agents such asamitriptyline, amoxapine, desipramine, doxepin, imipramine,nortriptyline, protriptyline, trimipramine; contraceptives (e.g.,norethindrone combinations, such as with ethinyl estradiol or withmestranol); and antinauseants/antiemetic agents (e.g., dimenhydrinate,hydroxyzine, meclizine, metoclopramide, prochlorperazine, promethazine,scopolamine, thiethylperazine, triethobenzamide).

Other therapeutic agents that can likewise be utilized within thepresent invention include antiasthmatic agents, antipsychotic agents,bronchodilators, gold compounds, hypoglycemic agents, hypolipedemicagents, anesthetics, vaccines, agents which affect bone metabolism,anti-diarrhetics, fertility agents, muscle relaxants, appetitesuppressants, hormones such as thyroid hormone, estrogen, progesterone,cortisone and/or growth hormone, other biologically active moleculessuch as insulin, as well as T_(H)1 (e.g., Interleukins-2, -12, and -15,gamma interferon) or T_(H)2 (e.g., Interleukins-4 and -10) cytokines.

Although the above therapeutic agents have been provided for thepurposes of illustration, it should be understood that the presentinvention is not so limited. For example, although agents arespecifically referred to above, the present invention should beunderstood to include analogues, derivatives and conjugates of suchagents. As an illustration, paclitaxel should be understood to refer tonot only the common chemically available form of paclitaxel, butanalogues (e.g., taxotere, as noted above) and paclitaxel conjugates(e.g., paclitaxel-PEG, paclitaxel-dextran, or paclitaxel-xylos). Inaddition, as will be evident to one of skill in the art, although theagents set forth above may be noted within the context of one class,many of the agents listed in fact have multiple biological activities.Further, more than one therapeutic agent may be utilized at a time(i.e., in combination), or delivered sequentially.

Polymeric Carriers

As noted above, therapeutic compositions of the present invention mayadditionally comprise a polymeric carrier. A wide variety of polymericcarriers may be utilized to contain and or delivery one or more of thetherapeutic agents discussed above, including for example bothbiodegradable and non-biodegradable compositions. Representativeexamples of biodegradable compositions include albumin, collagen,gelatin, starch, cellulose (methylcellulose, hydroxypropylcellulose,hydroxypropylmethylcellulose, carboxymethylcellulose, cellulose acetatephthalate, cellulose acetate succinate, hydroxypropylmethylcellulosephthalate), casein, dextrans, polysaccharides, fibrinogen, poly(D,Llactide), poly(D,L-lactide-co-glycolide), poly(glycolide),poly(hydroxybutyrate), poly(alkylcarbonate) and poly(orthoesters),polyesters, poly(hydroxyvaleric acid), polydioxanone, poly(ethyleneterephthalate), poly(malic acid), poly(tartronic acid), polyanhydrides,polyphosphazenes, poly(amino acids and their copolymers (see generallyIllum, L., Davids, S. S. (eds.) “Polymers in controlled Drug Delivery”Wright, Bristol, 1987; Arshady, J. Controlled Release 17:1-22, 1991;Pitt, Int. J. Phar. 59:173-196, 1990; Holland et al., J. ControlledRelease 4:155-0180, 1986). Representative examples of nondegradablepolymers include EVA copolymers, silicone rubber, acrylic polymers(polyacrylic acid, polymethylacrylic acid, polymethylmethacrylate,polyalkylcynoacrylate), polyethylene, polyproplene, polyamides (nylon6,6), polyurathane, poly(ester urathanes), poly(ether urathanes),poly(ester-urea), polyethers (poly(ethylene oxide), poly(propyleneoxide), pluronics, poly(tetramethylene glycol))xxx,silicone rubbers andvinyl polymers [polyvinylpyrrolidone, poly(vinyl alcohol, poly(vinylacetate phthalate. Polymers may also be developed which are eitheranionic (e.g., alginate, carrageenin, caboxymethyl cellulose andpoly(acrylic acid), or cationic (e.g., Chitosan, poly-1-lysine,polyethylenimine, and poly (allyl amine)) (see generally, Dunn et al.,J. Applied Polymer Sci. 50:353-365, 1993; Cascone et al., J. MaterialsSci.: Materials in Medicine 5:770-774, 1994; Shiraishi et al., Biol.Pharm. Bull. 16(11):1164-1168, 1993; Thacharodi and Rao, Int'l J. Pharm.120:115-118, 1995; Miyazaki et al., Int'l J. Pharm. 118:257-263, 1995).Particularly preferred polymeric carriers include poly(ethylene-vinylacetate) (40% cross-linked), poly (D,L-lactic acid) oligomers andpolymers, poly (L-lactic acid) oligomers and polymers, poly (glycolicacid), copolymers of lactic acid and glycolic acid, poly (caprolactone),poly (valerolactone), polyanhydrides, copolymers of poly (caprolactone)or poly (lactic acid) with polyethylene glycol and blends thereof.

Polymeric carriers can be fashioned in a variety of forms, with desiredrelease characteristics and/or with specific desired properties. Forexample, polymeric carriers may be fashioned to release a therapeuticagent upon exposure to a specific triggering event such as pH (see,e.g., Heller et al., “Chemically Self-Regulated Drug Delivery Systems,”in Polymers in Medicine III, Elsevier Science Publishers B.V.,Amsterdam, 1988, pp. 175-188; Kang et al., J. Applied Polymer Sci.48:343-354, 1993; Dong et al., J. Controlled Release 19:171-178, 1992;Dong and Hoffman, J. Controlled Release 15:141-152, 1991; Kim et al., J.Controlled Release 28:143-152, 1994; Cornejo-Bravo et al., J. ControlledRelease 33:223-229, 1995; Wu and Lee, Pharm. Res. 10(10):1544-1547,1993; Serres et al., Pharm. Res. 13(2):196-201, 1996; Peppas,“Fundamentals of pH- and Temperature-Sensitive Delivery Systems,” inGurny et al. (eds.), Pulsatile Drug Delivery, WissenschaftlicheVerlagsgesellschaft mbH, Stuttgart, 1993, pp. 41-55; Doelker, “CelluloseDerivatives,” 1993, in Peppas and Langer (eds.), Biopolymers I,Springer-Verlag, Berlin). Representative examples of pH-senstivepolymers include poly(acrylic acid) and its derivatives (including forexample, homopolymers such as poly(aminocarboxylic acid); poly(acrylicacid); poly(methyl acrylic acid)), copolymers of such homopolymers, andcopolymers of poly(acrylic acid) and acrylmonomers such as thosediscussed above. Other pH sensitive polymers include polysaccharidessuch as cellulose acetate phthalate; hydroxypropylmethylcellulosephthalate; hydroxypropylmethylcellulose acetate succinate; celluloseacetate trimellilate; and chitosan. Yet other pH sensitive polymersinclude any mixture of a pH sensitive polymer and a water solublepolymer.

Likewise, polymeric carriers can be fashioned which are temperaturesensitive (see, e.g., Chen et al., “Novel Hydrogels of aTemperature-Sensitive Pluronic Grafted to a Bioadhesive Polyacrylic AcidBackbone for Vaginal Drug Delivery,” in Proceed. Intern. Symp. Control.Rel. Bioact. Mater. 22:167-168, Controlled Release Society, Inc., 1995;Okano, “Molecular Design of Stimuli-Responsive Hydrogels for TemporalControlled Drug Delivery,” in Proceed. Intern. Symp. Control. Rel.Bioact. Mater. 22:111-112, Controlled Release Society, Inc., 1995;Johnston et al., Pharm. Res. 9(3):425-433, 1992; Tung, Int'l J. Pharm.107:85-90, 1994; Harsh and Gehrke, J. Controlled Release 17:175-186,1991; Bae et al., Pharm. Res. 8(4):531-537, 1991; Dinarvand andD'Emanuele, J. Controlled Release 36:221-227, 1995; Yu and Grainger,“Novel Thermo-sensitive Amphiphilic Gels: PolyN-isopropylacrylamide-co-sodium acrylate-co-n-N-alkylacrylamide NetworkSynthesis and Physicochemical Characterization,” Dept. of Chemical &Bioligal Sci., Oregon Graduate Institute of Science & Technology,Beaverton, Oreg., pp. 820-821; Zhou and Smid, “Physical Hydrogels ofAssociative Star Polymers,” Polymer Research Institute, Dept. ofChemistry, College of Environmental Science and Forestry, State Univ. ofNew York, Syracuse, N.Y., pp. 822-823; Hoffman et al., “CharacterizingPore Sizes and Water ‘Structure’ in Stimuli-Responsive Hydrogels,”Center for Bioengineering, Univ. of Washington, Seattle, Wash., p. 828;Yu and Grainger, “Thermo-sensitive Swelling Behavior in CrosslinkedN-isopropylacrylamide Networks: Cationic, Anionic and AmpholyticHydrogels,” Dept. of Chemical & Biological Sci., Oregon GraduateInstitute of Science & Technology, Beaverton, Oreg., pp. 829-830; Kim etal., Pharm. Res. 9(3):283-290, 1992; Bae et al., Pharm. Res.8(5):624-628, 1991; Kono et al., J. Controlled Release 30:69-75, 1994;Yoshida et al., J. Controlled Release 32:97-102, 1994; Okano et al., J.Controlled Release 36:125-133, 1995; Chun and Kim, J. Controlled Release38:39-47, 1996; D'Emanuele and Dinarvand, Int'l J. Pharm. 118:237-242,1995; Katono et al., J. Controlled Release 16:215-228, 1991; Hoffman,“Thermally Reversible Hydrogels Containing Biologically Active Species,”in Migliaresi et al. (eds.), Polymers in Medicine III, Elsevier SciencePublishers B.V., Amsterdam, 1988, pp. 161-167; Hoffman, “Applications ofThermally Reversible Polymers and Hydrogels in Therapeutics andDiagnostics,” in Third International Symposium on Recent Advances inDrug Delivery Systems, Salt Lake City, Utah, Feb. 24-27, 1987, pp.297-305; Gutowska et al., J. Controlled Release 22:95-104, 1992; Palasisand Gehrke, J. Controlled Release 18:1-12, 1992; Paavola et al., Pharm.Res. 12(12):1997-2002, 1995).

Representative examples of thermogelling polymers, and their gelatintemperature (LCST (° C.)) include homopolymers such aspoly(N-methyl-N-n-propylacrylamide), 19.8; poly(N-n-propylacrylamide),21.5; poly(N-methyl-N-isopropylacrylamide), 22.3;poly(N-n-propylmethacrylamide), 28.0; poly(N-isopropylacrylamide), 30.9;poly(N, n-diethylacrylamide), 32.0; poly(N-isopropylmethacrylamide),44.0; poly(N-cyclopropylacrylamide), 45.5; poly(N-ethylmethyacrylamide),50.0; poly(N-methyl-N-ethylacrylamide), 56.0;poly(N-cyclopropylmethacrylamide), 59.0; poly(N-ethylacrylamide), 72.0.Moreover thermogelling polymers may be made by preparing copolymersbetween (among) monomers of the above, or by combining such homopolymerswith other water soluble polymers such as acrylmonomers (e.g., acrylicacid and derivatives thereof such as methylacrylic acid, acrylate andderivatives thereof such as butyl methacrylate, acrylamide, andN-n-butyl acrylamide).

Other representative examples of thermogelling polymers includecellulose ether derivatives such as hydroxypropyl cellulose, 41° C.;methyl cellulose, 55° C.; hydroxypropylmethyl cellulose, 66° C.; andethylhydroxyethyl cellulose, and pluronics such as F-127, 10-15° C.;L-122, 19° C.; L-92, 26° C.; L-81, 20° C.; and L-61, 24° C.

A wide variety of forms may be fashioned by the polymeric carriers ofthe present invention, including for example, rod-shaped devices,pellets, slabs, or capsules (see, e.g., Goodell et al., Am. J. Hosp.Pharm. 43:1454-1461, 1986; Langer et al., “Controlled release ofmacromolecules from polymers”, in Biomedical polymers, Polymericmaterials and pharmaceuticals for biomedical use, Goldberg, E. P.,Nakagim, A. (eds.) Academic Press, pp. 113-137, 1980; Rhine et al., J.Pharm. Sci. 69:265-270, 1980; Brown et al., J. Pharm. Sci. 72:1181-1185,1983; and Bawa et al., J. Controlled Release 1:259-267, 1985).Therapeutic agents may be linked by occlusion in the matrices of thepolymer, bound by covalent linkages, or encapsulated in microcapsules.Within certain preferred embodiments of the invention, therapeuticcompositions are provided in non-capsular formulations such asmicrospheres (ranging from nanometers to micrometers in size), pastes,threads of various size, films and sprays.

Preferably, therapeutic compositions of the present invention arefashioned in a manner appropriate to the intended use. Within certainaspects of the present invention, the therapeutic composition should bebiocompatible, and release one or more therapeutic agents over a periodof several days to months. For example, “quick release” or “burst”therapeutic compositions are provided that release greater than 10%,20%, or 25% (w/v) of a therapeutic agent (e.g., paclitaxel) over aperiod of 7 to 10 days. Such “quick release” compositions should, withincertain embodiments, be capable of releasing chemotherapeutic levels(where applicable) of a desired agent. Within other embodiments, “lowrelease” therapeutic compositions are provided that release less than 1%(w/v) of a therapeutic agent over a period of 7 to 10 days. Further,therapeutic compositions of the present invention should preferably bestable for several months and capable of being produced and maintainedunder sterile conditions.

Within certain aspects of the present invention, therapeuticcompositions may be fashioned in any size ranging from 50 nm to 500 μm,depending upon the particular use. Alternatively, such compositions mayalso be readily applied as a “spray”, which solidifies into a film orcoating. Such sprays may be prepared from microspheres of a wide arrayof sizes, including for example, from 0.1 μm to 3 μm, from 10 μm to 30μm, and from 30 μm to 100 μm.

Therapeutic compositions of the present invention may also be preparedin a variety of “paste” or gel forms. For example, within one embodimentof the invention, therapeutic compositions are provided which are liquidat one temperature (e.g., temperature greater than 37° C., such as 40°C., 45° C., 50° C., 55° C. or 60° C.), and solid or semi-solid atanother temperature (e.g., ambient body temperature, or any temperaturelower than 37° C.). Such “thermopastes” may be readily made given thedisclosure provided herein.

Within yet other aspects of the invention, the therapeutic compositionsof the present invention may be formed as a film. Preferably, such filmsare generally less than 5, 4, 3, 2, or 1, mm thick, more preferably lessthan 0.75 mm or 0.5 mm thick, and most preferably less than 500 μm to100 μm thick. Such films are preferably flexible with a good tensilestrength (e.g., greater than 50, preferably greater than 100, and morepreferably greater than 150 or 200 N/cm²), good adhesive properties(i.e., readily adheres to moist or wet surfaces), and have controlledpermeability.

Within certain embodiments of the invention, the therapeuticcompositions may also comprise additional ingredients such assurfactants (e.g. pluronics such as F-127, L-122, L-92, L-81, and L-61).

Within further aspects of the present invention, polymeric carriers areprovided which are adapted to contain and release a hydrophobiccompound, the carrier containing the hydrophobic compound in combinationwith a carbohydrate, protein or polypeptide. Within certain embodiments,the polymeric carrier contains or comprises regions, pockets, orgranules of one or more hydrophobic compounds. For example, within oneembodiment of the invention, hydrophobic compounds may be incorporatedwithin a matrix which contains the hydrophobic compound, followed byincorporation of the matrix within the polymeric carrier. A variety ofmatrices can be utilized in this regard, including for example,carbohydrates and polysaccharides such as starch, cellulose, dextran,methylcellulose, and hyaluronic acid, proteins or polypeptides such asalbumin, collagen and gelatin. Within alternative embodiments,hydrophobic compounds may be contained within a hydrophobic core, andthis core contained within a hydrophilic shell. For example, asdescribed within the Examples, paclitaxel may be incorporated into ahydrophobic core (e.g., of the poly D,L lactic acid-PEG or MePEGaggregate) which has a hydrophilic shell.

A wide variety of hydrophobic compounds may be released from thepolymeric carriers described above, including for example: certainhydrophobic compounds which disrupt microtubule function such aspaclitaxel and estramustine; hydrophobic proteins such as myelin basicprotein, proteolipid proteins of CNS myelin, hydrophobic cell wallprotein, porins, membrane proteins (EMBO J. 12(9):3409-3415, 1993),myelin oligodendrocyte glycoprotein (“MOG”) (Biochem. and Mol. Biol.Int. 30(5):945-958, 1993, P27 Cancer Res. 53(17):4096-4101, 1913,bacterioopsin, human surfactant protein (“HSB”; J. Biol. Chem.268(15):11160-11166, 1993), and SP-B or SP-C (Biochimica et BiophysicaActa 1105(1):161-169, 1992).

Representative examples of the incorporation of therapeutic agents suchas those described above into a polymeric carriers to form a therapeuticcomposition, is described in more detail below in the Examples.

Other Carriers

Other carriers that may likewise be utilized to contain and deliver thetherapeutic agents described herein include: hydroxypropyl βcyclodextrin (Cserhati and Hollo, Int. J. Pharm. 108:69-75, 1994),liposomes (see e.g., Sharma et al., Cancer Res. 53:5877-5881, 1993;Sharma and Straubinger, Pharm. Res. 1](60):889-896, 1994; WO 93/18751;U.S. Pat. No. 5,242,073), liposome/gel (WO 94/26254), nanocapsules(Bartoli et al., J. Microencapsulation 7(2):191-197, 1990), micelles(Alkan-Onyuksel et al., Pharm. Res. 11(2):206-212, 1994), implants(Jampel et al., Invest. Ophthalm. Vis. Science 34(11):3076-3083, 1993;Walter et al., Cancer Res. 54:22017-2212, 1994) nanoparticles(Violanteand Lanzafame PAACR), nanoparticles—modified (U.S. Pat. No. 5,145,684),nanoparticles (surface modified) (U.S. Pat. No. 5,399,363), taxolemulsion/solution (U.S. Pat. No. 5,407,683), micelle (surfactant) (U.S.Pat. No. 5,403,858), synthetic phospholipid compounds (U.S. Pat. No.4,534,899), gas borne dispersion (U.S. Pat. No. 5,301,664), liquidemulsions, foam spray, gel lotion cream, ointment, dispersed vesicles,particles or droplets solid- or liquid-aerosols, microemulsions (U.S.Pat. No. 5,330,756), polymeric shell (nano- and micro-capsule) (U.S.Pat. No. 5,439,686), taxoid-based compositions in a surface-active agent(U.S. Pat. No. 5,438,072), emulsion (Tarr et al., Pharm Res. 4: 62-165,1987), nanospheres (Hagan et al., Proc. Intern. Symp. Control Rel.Bioact. Mater. 22, 1995; Kwon et al., Pharm Res. 12(2):192-195; Kwon etal., Pharm Res. 10(7):970-974; Yokoyama et al., J. Contr. Rel.32:269-277, 1994; Gref et al., Science 263:1600-1603, 1994; Bazile etal., J. Pharm. Sci. 84:493-498, 1994) and implants (U.S. Pat. No.4,882,168).

As discussed in more detail below, therapeutic agents of the presentinvention, which are optionally incorporated within one of the carriersdescribed herein to form a therapeutic composition, may be prepared andutilized to treat or prevent a wide variety of diseases.

Treatment or Prevention of Disease

As noted above, the present invention provides methods for treating orpreventing a wide variety of diseases associated with the obstruction ofbody passageways, including for example, vascular diseases, neoplasticobstructions, inflammatory diseases, and infectious diseases.

For example, within one aspect of the present invention a wide varietyof therapeutic compositions as described herein may be utilized to treatvascular diseases that cause obstruction of the vascular system.Representative examples of such diseases include artherosclerosis of allvessels (around any artery, vein or graft) including, but not restrictedto: the coronary arteries, aorta, iliac arteries, carotid arteries,common femoral arteries, superficial femoral arteries, poplitealarteries, and at the site of graft anastomosis; vasospasms (e.g,coronary vasospasms and Raynaud's Disease); restenosis (obstruction of avessel at the site of a previous intervention such as balloonangioplasty, bypass surgery, stent insertion and graft insertion);inflammatory and autoimmune conditions (e.g. Temporal Arteritis,vasculitis).

Briefly, in vascular diseases such as atherosclerosis, white cells,specifically monocytes and T lymphocytes adhere to endothelial cells,especially at locations of arterial branching. After adhering to theendothelium, leukocytes migrate across the endothelial cell lining inresponse to chemostatic stimuli, and accumulate in the intima of thearterial wall, along with smooth muscle cells. This initial lesion ofathersosclerosis development is known as the “fatty streak”. Monocyteswithin the fatty streak differentiate into macrophages; and themacrophages and smooth muscle cells progressively take up lipids andlipoprotein to become foam cells.

As macrophages accumulate, the overlying endothelium becomesmechanically disrupted and chemically altered by oxidized lipid,oxygen-derived free radicals and proteases which are released bymacrophages. Foam cells erode through the endothelial surface causingmicro-ulcerations of the vascular wall. Exposure of potentiallythrombogenic subendothelial tissues (such as collagen and otherproteins) to components of the bloodstream results in adherence ofplatelets to regions of disrupted endothelium. Platelet adherence andother events triggers the elaboration and release of growth factors intothis mileau, including platelet-derived growth factor (PDGF), plateletactivating factor (PAF), and interleukins 1 and 6 (IL-1, IL-6). Theseparacrine factors are thought to stimulate vascular smooth muscle cell(VSMC) migration and proliferation.

In the normal (non-diseased) blood vessel wall, vascular smooth musclecells have a contractile phenotype and low index of mitotic activity.However, under the influence of cytokines and growth factors released byplatelets, macrophages and endothelial cells, VSMC undergo phenotypicalteration from mature contractile cells to immature secretory cells.The transformed VSMC proliferate in the media of the blood vessel wall,migrate into the intima, continue to proliferate in the intima andgenerate large quantities of extracellular matrix. This transforms theevolving vascular lesion into a fibrous plaque. The extracellular matrixelaborated by secretory VSMC includes collagen, elastin, glycoproteinand glycosaminoglycans, with collagen comprising the major extracellularmatrix component of the atherosclerotic plaque. Elastin andglycosaminoglycans bind lipoproteins and also contribute to lesiongrowth. The fibrous plaque consists of a fibrous cap of dense connectivetissue of varying thickness containing smooth muscle cells and overlyingmacrophages, T cells and extracellular material.

In addition to PDGF, IL-1 and IL-6, other mitogenic factors are producedby cells which infiltrate the vessel wall including: transforming growthfactor beta (TGF-β), fibroblast growth factor (FGF), thrombospondin,serotonin, thromboxane A₂, norepenephrine, and angiotension II. Thisresults in the recruitment of more cells, elaboration of furtherextracellular matrix and the accumulation of additional lipid. Thisprogressively enlarges the atherosclerotic lesion until it significantlyencroaches upon the vascular lumen. Initially, obstructed blood flowthrough the vascular tube causes ischemia of the tissues distal to theatherosclerotic plaque only when increased flow is required—later as thelesion further blocks the artery, ischemia occurs at rest.

Macrophages in the enlarging atherosclerotic plaque release oxidizedlipid, free radicals, elastases, and collageneses that cause cell injuryand necrosis of neighbouring tissues. The lesion develops a necroticcore and is transformed into a complex plaque. Complex plaques areunstable lesions that can: break off causing embolization; localhemorrhage (secondary to rupture of the vasa vasora supplying the plaquewhich results in lumen obstruction due to rapid expansion of thelesion); or ulceration and fissure formation (this exposes thethrombogenic necrotic core to the blood stream producing localthrombosis or distal embolization). Even should none of the abovesequela occur, the adherent thrombus may become organized andincorporated into the plaque, thereby accelerating its growth.Furthermore, as the local concentrations of fibrinogen and thrombinincrease, proliferation of vascular smooth muscle cells within the mediaand intima is stimulated; a process which also ultimately leads toadditional narrowing of the vessel.

The intima and media of normal arteries are oxygenated and supplied withnutrition from the lumen of the artery or from the vasa vasorum in theadventitia. With the development of atherosclerotic plaque, microvesselsarising from the adventitial vasa vasorum extend into the thickenedintima and media. This vascular network becomes more extensive as theplaque worsens and diminishes with plaque regression.

Hemorrhage from these microvessels may precipitate sudden expansion andrupture of plaque in association with arterial dissection, ulceration,or thrombosis. It has also been postulated that the leakage of plasmaproteins from these microvessels may attract inflammatory infiltratesinto the region and these inflammatory cells may contribute to the rapidgrowth of atherosclerotic plaque and to associated complications(through local edema and inflammation).

In order to treat vascular diseases, such as those discussed above, awide variety of therapeutic agents (either with or without a carrier)may be delivered to the external portion of the body passageway, or tosmooth muscle cells via the adventia of the body passageway.Particularly preferred therapeutic agents in this regard includeanti-angiogenic factors, inhibitors of platelet adhesion/aggregation(e.g., aspirin, dipyridamole, thromboxane synthesis inhibitors, fishoils that result in production of thromboxane AE rather than the morepotent thromboxane A2, antibodies against the platelet IIb/IIIareceptors that binds fibrinogen and prostacyclin), vasodilators (e.g.,calcium entry blockers, and the nitric oxide donors nitroglycerine,nitroprusside, and molsidomine) and anthithrombotics and thrombinantagonists (e.g., heparin (low-molecular-weight heparins, warfarinandudin). Other therapeutics which may be utilized includeanti-inflammatory agents (e.g., glucorticoids, dexamethasone andmethylprednisolone), growth factor inhibitors (e.g., PDGF antagonistsuch as trapidil; receptor inhibitors (e.g., inhibitors of the receptorsfor FGF, VEGF, PDGF and TNF), including inhibitors of tyrosine kinaseand promoters of tyrosine phosphatase; somatostatin analogs, includingangiopeptin; angiotensin converting enzyme inhibitors; and 5HT₂serotenergic receptor antagonists such as ketanserin). Yet othertherapeutic agents include anti-proliferative agents (e.g., colchicine,heparin, beta (e.g., P-32) or gamma emitters (e.g., Ir-192),calcium-entry blockers such as verapamil, diltiazem and nifedipine,cholesterol-lowering HMB Co-A reductase inhibitors such as lovastatin,compounds which disrupt microtubule function such as paclitaxel andnitric oxide donors as discussed above), and promoters ofre-endothelialization (e.g., bFGF and vascular endothelial cell growthfactor).

Within other aspects of the invention, the therapeutic agents orcompositions described herein may be utilized to treat neoplasticobstructions. Briefly, as utilized herein, a “neoplastic obstruction”should be understood to include any neoplastic (benign or malignant)obstruction of a bodily tube regardless of tube location or histologicaltype of malignancy present. Representative examples includegastrointestinal diseases (e.g., oral-pharyngeal carcinoma(adenocarcinoma, esophageal carcinoma (squamous cell, adenocarcinoma,lymphoma, melanoma), gastric carcinoma (adenocarcinoma, linitisplastica, lymphoma, leiomyosarcoma), small bowel tumors (adenomas,leiomyomas, lipomas, adenocarcinomas, lymphomas, carcinoid tumors),colon cancer (adenocarcinoma) and anorectal cancer); biliary tractdiseases (e.g., neoplasms resulting in biliary obstruction such aspancreatic carcinoma (ductal adenocarcinoma, islet cell tumors,cystadenocarcinoma), cholangiocarcinoma and hepatocellular carcinoma);pulmonary diseases (e.g., carcinoma of the lung and/ortracheal/bronchial passageways (small cell lung cancer, non-small celllung cancer); female reproductive diseases (e.g., malignancies of thefallopian tubes, uterine cancer, cervical cancer, vaginal cancer); malereproductive diseases (e.g,. testicular cancer, cancer of theepididymus, tumors of the vas deferens, prostatic cancer, benignprostatic hypertrophy); and urinary tract diseases (e.g., renal cellcarcinoma, tumors of the renal pelvis, tumors of the urinary collectionsystem such as transitional cell carcinoma, bladder carcinoma, andurethral obstructions due to benign strictures, or malignancy).

As an example, benign prostatic hyperplasia (BPH) is the enlargement ofthe prostate, particularly the central portion of the gland whichsurrounds the urethra, which occurs in response to prolonged androgenicstimulation. It affects more than 80% of the men over 50 years of age.This enlargement can result in compression of the portion of the urethrawhich runs through the prostate, resulting in bladder outflow tractobstruction, i.e., an abnormally high bladder pressure is required togenerate urinary flow. In 1980, 367,000 transurethral resections of theprostate were performed in the United States as treatment for BPH. Othertreatments include medication, transurethral sphincterotomy,transurethral laser or microwave, transurethral hyperthermia,transurethral ultrasound, transrectal microwave, transrectalhyperthermia, transrectal ultrasound and surgical removal. All havedisadvantages including interruption of the sphincter mechanismresulting in incontinence and stricture formation.

In order to treat neoplastic diseases, such as those discussed above, awide variety of therapeutic agents (either with or without a polymericcarrier) may be delivered to the external portion of the bodypassageway, or to smooth muscle cells via the adventia of the bodypassageway. Particularly preferred therapeutic agents in this regardinclude anti-angiogenic, anti-proliferative or anti-neoplastic agentsdiscussed above, including for example, compounds which disruptmicrotuble function, such as paclitaxel and derivatives or analoguesthereof.

For example, within one preferred embodiment a needle or catheter isguided into the prostate gland adjacent to the urethra via thetransrectal route (or alternatively transperineally) under ultrasoundguidance and through this deliver a therapeutic agent, preferably inseveral quadrants of the gland, particularly around the urethra. Theneedle or catheter can also be placed under direct palpation or underendoscopic, fluoroscopic, CT or MRI guidance, and administered atintervals. As an alternative, the placement of pellets via a catheter ortrocar can also be accomplished. The above procedures can beaccomplished alone or in conjunction with a stent placed in theprostatic urethra. By avoiding urethral instrumentation or damage to theurethra, the sphincter mechanism would be left intact, avoidingincontinence, and a stricture is less likely.

Within other aspects of the invention, methods are provided forpreventing or treating inflammatory diseases which affect or cause theobstruction of a body passageway. Inflammatory diseases include bothacute and chronic inflammation which result in obstruction of a varietyof body tubes. Representative examples include vasculitis (e.g., Giantcell arteritis (temporal arteritis, Takayasu's arteritis), polyarteritisnodosa, allergic angiitis and granulomatosis (Churg-Strauss disease),polyangiitis overlap syndrome, hypersensitivity vasculitis(Henoch-Schonlein purpura), serum sickness, drug-induced vasculitis,infectious vasculitis, neoplastic vasculitis, vasculitis associated withconnective tissue disorders, vasculitis associated with congenitaldeficiencies of the complement system), Wegener's granulomatosis,Kawasaki's disease, vasculitis of the central nervous system, Buerger'sdisease and systemic sclerosis); gastrointestinal tract diseases (e.g.,pancreatitis, Crohn's Disease, Ulcerative Colitis, Ulcerative Proctitis,Primary Sclerosing Cholangitis, benign strictures of any cause includingideopathic (e.g., strictures of bile ducts, esophagus, duodenum, smallbowel or colon)); respiratory tract diseases (e.g, asthma,hypersensitivity pneumonitis, asbestosis, silicosis, and other forms ofpneumoconiosis, chronic bronchitis and chronic obstructive airwaydisease); nasolacrimal duct diseases (e.g., strictures of all causesincluding ideopathic); and eustachean tube diseases (e.g., strictures ofall causes including ideopathic).

In order to treat inflammatory diseases, such as those discussed above,a wide variety of therapeutic agents (either with or without a carrier)may be delivered to the external portion of the body passageway, or tosmooth muscle cells via the adventia of the body passageway.Particularly preferred therapeutic agents in this regard include bothnonsteroidal agents (“NSAIDS”) and steroidal agents, as well as theanti-angiogenic factors discussed above. Other agents which may also beutilized include a wide variety of anti-angiogenic facts, including forexample compounds which disrupt microtubule function, such aspaclitaxel, and lighter “d” group transition metals.

Within yet other aspects of the present invention, methods are providedfor treating or preventing infectious diseases that are associated with,or causative of, the obstruction of a body passageway. Briefly,infectious diseases include several acute and chronic infectiousprocesses can result in obstruction of body passageways including forexample, obstructions of the male reproductive tract (e.g., stricturesdue to urethritis, epididymitis, prostatitis); obstructions of thefemale reproductive tract (e.g., vaginitis, cervicitis, pelvicinflammatory disease (e.g., tuberculosis, gonococcus, chlamydia,enterococcus and syphilis); urinary tract obstructions (e.g., cystitis,urethritis); respiratory tract obstructions (e.g., chronic bronchitis,tuberculosis, other mycobacterial infections (MAI, etc.), anaerobicinfections, fungal infections and parasitic infections) andcardiovascular obstructions (e.g., mycotic aneurysms and infectiveendocarditis).

In order to treat infectious diseases, such as those discussed above, awide variety of therapeutic agents (either with or without a carrier)may be delivered to the external portion of the body passageway, or tosmooth muscle cells via the adventia of the body passageway.Particularly preferred therapeutic agents in this regard include a widevariety of antibiotics as discussed above.

Formulation and Administration

As noted above, therapeutic compositions of the present invention may beformulated in a variety of forms (e.g., microspheres, pastes, films orsprays). Further, the compositions of the present invention may beformulated to contain more than one therapeutic agents, to contain avariety of additional compounds, to have certain physical properties(e.g., elasticity, a particular melting point, or a specified releaserate). Within certain embodiments of the invention, compositions may becombined in order to achieve a desired effect (e.g., severalpreparations of microspheres may be combined in order to achieve both aquick and a slow or prolonged release of one or more anti-angiogenicfactor).

Therapeutic agents and compositions of the present invention may beadministered either alone, or in combination with pharmaceutically orphysiologically acceptable carrier, excipients or diluents. Generally,such carriers should be nontoxic to recipients at the dosages andconcentrations employed. Ordinarily, the preparation of suchcompositions entails combining the therapeutic agent with buffers,antioxidants such as ascorbic acid, low molecular weight (less thanabout 10 residues) polypeptides, proteins, amino acids, carbohydratesincluding glucose, sucrose or dextrins, chelating agents such as EDTA,glutathione and other stabilizers and excipients. Neutral bufferedsaline or saline mixed with nonspecific serum albumin are exemplaryappropriate diluents.

As noted above, therapeutic agents, therapeutic compositions, orpharmaceutical compositions provided herein may be prepared foradministration by a variety of different routes, including for example,directly to a body passageway under direct vision (e.g., at the time ofsurgery or via endoscopic procedures) or via percutaneous drug deliveryto the exterior (adventitial) surface of the body passageway (e.g.,perivascular delivery). Other representative routes of administrationinclude gastroscopy, ECRP and colonoscopy, which do not require fulloperating procedures and hospitalization, but may require the presenceof medical personnel.

Briefly, perivascular drug delivery involves percutaneous administrationof localized (often sustained release) therapeutic formulations using aneedle or catheter directed via ultrasound, CT, fluoroscopic, MRI orendoscopic guidance to the disease site. Alternatively the procedure canbe performed intra-operatively under direct vision or with additionalimaging guidance. Such a procedure can also be performed in conjunctionwith endovascular procedures such as angioplasty, atherectomy, orstenting or in association with an operative arterial procedure such asendarterectomy, vessel or graft repair or graft insertion.

For example, within one embodiment, polymeric paclitaxel formulationscan be injected into the vascular wall or applied to the adventitialsurface allowing drug concentrations to remain highest in regions wherebiological activity is most needed. This has the potential to reducelocal “washout” of the drug that can be accentuated by continuous bloodflow over the surface of an endovascular drug delivery device (such as adrug-coated stent). Administration of effective therapeutic agents tothe external surface of the vascular tube can reduce obstruction of thetube and reduce the risk of complications associated with intravascularmanipulations (such as restenosis, embolization, thrombosis, plaquerupture, and systemic drug toxicity).

For example, in a patient with narrowing of the superficial femoralartery, balloon angioplasty would be performed in the usual manner(i.e., passing a balloon angioplasty catheter down the artery over aguide wire and inflating the balloon across the lesion). Prior to, atthe time of, or after angioplasty, a needle would be inserted throughthe skin under ultrasound, fluoroscopic, or CT guidance and atherapeutic agent (e.g., paclitaxel impregnated into a slow releasepolymer) would be infiltrated through the needle or catheter in acircumferential manner directly around the area of narrowing in theartery. This could be performed around any artery, vein or graft, butideal candidates for this intervention include diseases of the carotid,coronary, iliac, common femoral, superficial femoral and poplitealarteries and at the site of graft anastomosis. Logical venous sitesinclude infiltration around veins in which indwelling catheters areinserted.

The therapeutic agents, therapeutic compositions and pharmaceuticalcompositions provided herein may be placed within containers, along withpackaging material which provides instructions regarding the use of suchmaterials. Generally, such instructions include a tangible expressiondescribing the reagent concentration, as well as within certainembodiments, relative amounts of excipient ingredients or diluents(e.g., water, saline or PBS) which may be necessary to reconstitute theanti-angiogenic factor, anti-angiogenic composition, or pharmaceuticalcomposition.

The following examples are offered by way of illustration, and not byway of limitation.

EXAMPLES Example 1 Manufacture of “Pastes”

As noted above, the present invention provides a variety ofpolymeric-containing drug compositions that may be utilized within avariety of clinical situations. For example, compositions may beproduced: (1) as a “thermopaste” that is applied to a desired site as afluid, and hardens to a solid of the desired shape at a specifiedtemperature (e.g., body temperature); (2) as a spray (i.e., “nanospray”)which may delivered to a desired site either directly or through aspecialized apparatus (e.g., endoscopy), and which subsequently hardensto a solid which adheres to the tissue to which it is applied; (3) as anadherent, pliable, resilient, drug-loaded-polymer film applied to adesired site either directly or through a specialized apparatus, andwhich preferably adheres to the site to which it is applied; and (4) asa fluid composed of a suspension of microspheres in an appropriatecarrier medium, which is applied to a desired site either directly orvia a specialized apparatus, and which leaves a layer of microspheres atthe application site. Representative examples of each of the aboveembodiments is set forth in more detail below.

A. Procedure for Producing Thermopaste

Reagents and equipment which are utilized within the followingexperiments include a sterile glass syringe (1 ml), Corning hotplate/stirrer, 20 ml glass scintillation vial, moulds (e.g., 50 μl DSCpan or 50 ml centrifuge tube cap inner portion), scalpel and tweezers,Polycaprolactone (“PCL”—mol wt 10,000 to 20,000; Polysciences,Warrington, Pa. USA), and Paclitaxel (Sigma grade 95% purity minimum).

Weigh 5.00 g of polycaprolactone directly into a 20 ml glassscintillation vial. Place the vial in a 600 ml beaker containing 50 mlof water. Gently heat the beaker to 65° C. and hold it at thattemperature for 20 minutes. This allows the polymer to melt. Thoroughlymix a known weight of paclitaxel, or other angiogenesis inhibitor intothe melted polymer at 65° C. Pour the melted polymer into a prewarmed(60° C. oven) mould. Use a spatula to assist with the pouring process.Allow the mould to cool so the polymer solidifies. Cut or break thepolymer into small pieces (approximately 2 mm by 2 mm in size). Thesepieces must fit into a 1 ml glass syringe. Remove the plunger from the 1ml glass syringe (do not remove the cap from the tip) and place it on abalance. Zero the balance.

Weigh 0.5 g of the pieces directly into the open end of the syringe.Place the glass syringe upright (capped tip downwards) into a 500 mlglass beaker containing distilled water at 65° C. (coming hot plate) sothat no water enters the barrel. The polymer melts completely within 10minutes in this apparatus. When the polymer pieces have melted, removethe barrel from the water bath, hold it horizontally and remove the cap.Insert the plunger into the barrel and compress the melted polymer intoa sticky mass at the tip end of the barrel. Cap the syringe and allow itto cool to room temperature.

For application, the syringe may be reheated to 60° C. and administeredas a liquid which solidifies when cooled to body temperature.

B. Procedure for Producing Nanospray

Nanospray is a suspension of small microspheres in saline. If themicrospheres are very small (i.e., under 1 μm in diameter) they form acolloid so that the suspension will not sediment under gravity. As isdescribed in more detail below, a suspension of 0.1 μm to 1 μmmicroparticles may be created suitable for deposition onto tissuethrough a finger pumped aerosol. Equipment and materials which may beutilized to produce nanospray include 200 ml water jacketed beaker(Kimax or Pyrex), Haake circulating water bath, overhead stirrer andcontroller with 2 inch diameter (4 blade, propeller type stainless steelstirrer; Fisher brand), 500 ml glass beaker, hot plate/stirrer (Corningbrand), 4×50 ml polypropylene centrifuge tubes (Nalgene), glassscintillation vials with plastic insert caps, table top centrifuge(Beckman), high speed centrifuge—floor model (JS 21 Beckman), Mettleranalytical balance (AJ 100, 0.1 mg), Mettler digital top loading balance(AE 163, 0.01 mg), automatic pipetter (Gilson), sterile pipette tips,pump action aerosol (Pfeiffer pharmaceuticals) 20 ml, laminar flow hood,Polycaprolactone (“PCL”—mol wt 10,000 to 20,000; Polysciences,Warrington, Pa. USA), “washed” (see previous) Ethylene Vinyl Acetate(“EVA”), Poly(DL)lactic acid (“PLA” mol wt 15,000 to 25,000;Polysciences), Polyvinyl Alcohol (“PVA”—mol wt 124,000 to 186,000; 99%hydrolyzed; Aldrich Chemical Co., Milwaukee, Wis. USA), Dichloromethane(“DCM” or “methylene chloride;” HPLC grade Fisher scientific), Distilledwater, sterile saline (Becton and Dickenson or equivalent)

1. Preparation of 5% (w/v) Polymer Solutions

Depending on the polymer solution being prepared, weigh 1.00 g of PCL orPLA or 0.50 g each of PLA and washed EVA directly into a 20 ml glassscintillation vial. Using a measuring cylinder, add 20 ml of DCM andtightly cap the vial. Leave the vial at room temperature (25° C.) forone hour or until all the polymer has dissolved (occasional hand shakingmay be used). Dissolving of the polymer can be determined by a visualcheck; the solution should be clear. Label the vial with the name of thesolution and the date it was produced. Store the solutions at roomtemperature and use within two weeks.

2. Preparation of 3.5% (w/v) Stock Solution of PVA

The solution can be prepared by following the procedure given below, orby diluting the 5% (w/v) PVA stock solution prepared for production ofmicrospheres (see Example 2). Briefly, 17.5 g of PVA is weighed directlyinto a 600 ml glass beaker, and 500 ml of distilled water is added.Place a 3 inch Teflon coated stir bar in the beaker. Cover the beakerwith a cover glass to reduce evaporation losses. Place the beaker in a2000 ml glass beaker containing 300 ml of water. This will act as awater bath. Stir the PVA at 300 rpm at 85° C. (Corning hotplate/stirrer) for 2 hours or until fully dissolved. Dissolving of thePVA can be determined by a visual check; the solution should be clear.Use a pipette to transfer the solution to a glass screw top storagecontainer and store at 4° C. for a maximum of two months. This solutionshould be warmed to room temperature before use or dilution.

3. Procedure for Producing Nanospray

Place the stirring assembly in a fume hood. Place 100 ml of the 3.5% PVAsolution in the 200 ml water jacketed beaker. Connect the Haake waterbath to this beaker and allow the contents to equilibrate at 27° C.(+/−1° C.) for 10 minutes. Set the start speed of the overhead stirrerat 3000 rpm (+/−200 rpm). Place the blade of the overhead stirrer halfway down in the PVA solution and start the stirrer. Drip 10 ml ofpolymer solution (polymer solution used based on type of nanospray beingproduced) into the stirring PVA over a period of 2 minutes using a 5 mlautomatic pipetter. After 3 minutes, adjust the stir speed to 2500 rpm(+/−200 rpm) and leave the assembly for 2.5 hours. After 2.5 hours,remove the stirring blade from the nanospray preparation and rinse with10 ml of distilled water. Allow the rinse solution to go into thenanospray preparation.

Pour the microsphere preparation into a 500 ml beaker. Wash the jacketedwater bath with 70 ml of distilled water. Allow the 70 ml rinse solutionto go into the microsphere preparation. Stir the 180 ml microspherepreparation with a glass rod and pour equal amounts of it into fourpolypropylene 50 ml centrifuge tubes. Cap the tubes. Centrifuge thecapped tubes at 10 000 g (+/−1000 g) for 10 minutes. Using a 5 mlautomatic pipetter or vacuum suction, draw 45 ml of the PVA solution offof each microsphere pellet and discard it. Add 5 ml of distilled waterto each centrifuge tube and use a vortex to resuspend the microspheresin each tube. Using 20 ml of distilled water, pool the four microspheresuspensions into one centrifuge tube. To wash the microspheres,centrifuge the nanospray preparation for 10 minutes at 10 000 g (+/−1000g). Draw the supernatant off of the microsphere pellet. Add 40 ml ofdistilled water and use a vortex to resuspend the microspheres. Repeatthis process two more times for a total of three washes. Do a fourthwash but use only 10 ml (not 40 ml) of distilled water when resuspendingthe microspheres. After the fourth wash, transfer the microspherepreparation into a preweighed glass scintillation vial.

Cap the vial and let it to sit for 1 hour at room temperature (25° C.)to allow the 2 μm and 3 μm diameter microspheres to sediment out undergravity. After 1 hour, draw off the top 9 ml of suspension using a 5 mlautomatic pipetter. Place the 9 ml into a sterile capped 50 mlcentrifuge tube. Centrifuge the suspension at 10 000 g (+/−1000 g) for10 minutes. Discard the supernatant and resuspend the pellet in 20 ml ofsterile saline. Centrifuge the suspension at 10 000 g (+/−1000 g) for 10minutes. Discard the supernatant and resuspend the pellet in sterilesaline. The quantity of saline used is dependent on the final requiredsuspension concentration (usually 10% w/v). Thoroughly rinse the aerosolapparatus in sterile saline and add the nanospray suspension to theaerosol.

C. Manufacture of Paclitaxel Loaded Nanospray

To manufacture nanospray containing paclitaxel, use Paclitaxel (Sigmagrade 95% purity). To prepare the polymer drug stock solution, weigh theappropriate amount of paclitaxel directly into a 20 ml glassscintillation vial. The appropriate amount is determined based on thepercentage of paclitaxel to be in the nanospray. For example, ifnanospray containing 5% paclitaxel was required, then the amount ofpaclitaxel weighed would be 25 mg since the amount of polymer added is10 ml of a 5% polymer in DCM solution (see next step).

Add 10 ml of the appropriate 5% polymer solution to the vial containingthe paclitaxel. Cap the vial and vortex or hand swirl it to dissolve thepaclitaxel (visual check to ensure paclitaxel dissolved). Label the vialwith the date it was produced. This is to be used the day it isproduced.

Follow the procedures as described above, except that polymer/drug(e.g., paclitaxel) stock solution is substituted for the polymersolution.

D. Procedure for Producing Film

The term film refers to a polymer formed into one of many geometricshapes. The film may be a thin, elastic sheet of polymer or a 2 mm thickdisc of polymer. This film is designed to be placed on exposed tissue sothat any encapsulated drug is released from the polymer over a longperiod of time at the tissue site. Films may be made by severalprocesses, including for example, by casting, and by spraying.

In the casting technique, polymer is either melted and poured into ashape or dissolved in dichloromethane and poured into a shape. Thepolymer then either solidifies as it cools or solidifies as the solventevaporates, respectively. In the spraying technique, the polymer isdissolved in solvent and sprayed onto glass, as the solvent evaporatesthe polymer solidifies on the glass. Repeated spraying enables a buildup of polymer into a film that can be peeled from the glass.

Reagents and equipment which were utilized within these experimentsinclude a small beaker, Corning hot plate stirrer, casting moulds (e.g.,50 ml centrifuge tube caps) and mould holding apparatus, 20 ml glassscintillation vial with cap (Plastic insert type), TLC atomizer,Nitrogen gas tank, Polycaprolactone (“PCL”—mol wt 10,000 to 20,000;Polysciences), Paclitaxel (Sigma 95% purity), Ethanol, “washed” (seeprevious) Ethylene vinyl acetate (“EVA”), Poly(DL)lactic acid (“PLA”—molwt 15,000 to 25,000; Polysciences), Dichloromethane (HPLC grade FisherScientific).

1. Procedure for Producing Films—Melt Casting

Weigh a known weight of PCL directly into a small glass beaker. Placethe beaker in a larger beaker containing water (to act as a water bath)and put it on the hot plate at 70° C. for 15 minutes or until thepolymer has fully melted. Add a known weight of drug to the meltedpolymer and stir the mixture thoroughly. To aid dispersion of the drugin the melted PCL, the drug may be suspended/dissolved in a small volume(<10% of the volume of the melted PCL) of 100% ethanol. This ethanolsuspension is then mixed into the melted polymer. Pour the meltedpolymer into a mould and let it to cool. After cooling, store the filmin a container.

2. Procedure for Producing Films—Solvent Casting

Weigh a known weight of PCL directly into a 20 ml glass scintillationvial and add sufficient DCM to achieve a 10% w/v solution. Cap the vialand mix the solution. Add sufficient paclitaxel to the solution toachieve the desired final paclitaxel concentration. Use hand shaking orvortexing to dissolve the paclitaxel in the solution. Let the solutionsit for one hour (to diminish the presence of air bubbles) and then pourit slowly into a mould. The mould used is based on the shape required.Place the mould in the fume hood overnight. This will allow the DCM toevaporate. Either leave the film in the mould to store it or peel it outand store it in a sealed container.

3. Procedure for Producing Films—Sprayed

Weigh sufficient polymer directly into a 20 ml glass scintillation vialand add sufficient DCM to achieve a 2% w/v solution. Cap the vial andmix the solution to dissolve the polymer (hand shaking). Assemble themoulds in a vertical orientation in a suitable mould holding apparatusin the fume hood. Position this mould holding apparatus 6 to 12 inchesabove the fume hood floor on a suitable support (e.g., inverted 2000 mlglass beaker) to enable horizontal spraying. Using an automatic pipette,transfer a suitable volume (minimum 5 ml) of the 2% polymer solution toa separate 20 ml glass scintillation vial. Add sufficient paclitaxel tothe solution and dissolve it by hand shaking the capped vial. To preparefor spraying, remove the cap of this vial and dip the barrel (only) ofan TLC atomizer into the polymer solution. Note: the reservoir of theatomizer is not used in this procedure—the 20 ml glass vial acts as areservoir.

Connect the nitrogen tank to the gas inlet of the atomizer. Graduallyincrease the pressure until atomization and spraying begins. Note thepressure and use this pressure throughout the procedure. To spray themoulds use 5 second oscillating sprays with a 15 second dry time betweensprays. During the dry time, finger crimp the gas line to avoid wastageof the spray. Spraying is continued until a suitable thickness ofpolymer is deposited on the mould. The thickness is based on therequest. Leave the sprayed films attached to the moulds and store insealed containers.

E. Procedure for Producing Nanopaste

Nanopaste is a suspension of microspheres suspended in a hydrophilicgel. Within one aspect of the invention, the gel or paste can be smearedover tissue as a method of locating drug loaded microspheres close tothe target tissue. Being water based, the paste will soon become dilutedwith bodily fluids causing a decrease in the stickiness of the paste anda tendency of the microspheres to be deposited on nearby tissue. A poolof microsphere encapsulated drug is thereby located close to the targettissue.

Reagents and equipment which were utilized within these experimentsinclude glass beakers, Carbopol 925 (pharmaceutical grade, GoodyearChemical Co.), distilled water, sodium hydroxide (1 M) in watersolution, sodium hydroxide solution (5 M) in water solution,microspheres in the 0.1 lm to 3 lm size range suspended in water at 20%w/v (See previous).

1. Preparation of 5% w/v Carbopol Gel

Add a sufficient amount of carbopol to 1 M sodium hydroxide to achieve a5% w/v solution. To dissolve the carbopol in the 1 M sodium hydroxide,allow the mixture to sit for approximately one hour. During this timeperiod, stir the mixture using a glass rod. After one hour, take the pHof the mixture. A low pH indicates that the carbopol is not fullydissolved. The pH you want to achieve is 7.4. Use 5 M sodium hydroxideto adjust the pH. This is accomplished by slowly adding drops of 5 Msodium hydroxide to the mixture, stirring the mixture and taking the pHof the mixture. It usually takes approximately one hour to adjust the pHto 7.4. Once a pH of 7.4 is achieved, cover the gel and let it sit for 2to 3 hours. After this time period, check the pH to ensure it is stillat 7.4. If it has changed, adjust back to pH 7.4 using 5 M sodiumhydroxide. Allow the gel to sit for a few hours to ensure the pH isstable at 7.4. Repeat the process until the desired pH is achieved andis stable. Label the container with the name of the gel and the date.The gel is to be used to make nanopaste within the next week.

2. Procedure for Producing Nanopaste

Add sufficient 0.1 μm to 3 μm microspheres to water to produce a 20%suspension of the microspheres. Put 8 ml of the 5% w/v carbopol gel in aglass beaker. Add 2 ml of the 20% microsphere suspension to the beaker.Using a glass rod or a mixing spatula, stir the mixture to thoroughlydisperse the microspheres throughout the gel. This usually takes 30minutes. Once the microspheres are dispersed in the gel, place themixture in a storage jar. Store the jar at 4° C. It must be used withina one month period.

Example 2 Manufacture of Microspheres

Equipment which is preferred for the manufacture of microspheresdescribed below include: 200 ml water jacketed beaker (Kimax or Pyrex),Haake circulating water bath, overhead stirrer and controller with 2inch diameter (4 blade, propeller type stainless steel stirrer—Fisherbrand), 500 ml glass beaker, hot plate/stirrer (Corning brand), 4×50 mlpolypropylene centrifuge tubes (Nalgene), glass scintillation vials withplastic insert caps, table top centrifuge (GPR Beckman), high speedcentrifuge-floor model (JS 21 Beckman), Mettler analytical balance (AJ100, 0.1 mg), Mettler digital top loading balance (AE 163, 0.01 mg),automatic pipetter (Gilson). Reagents include Polycaprolactone(“PCL”—mol wt 10,000 to 20,000; Polysciences, Warrington Pa., USA),“washed” (see later method of “washing”) Ethylene Vinyl Acetate (“EVA”),Poly(DL)lactic acid (“PLA”—mol wt 15,000 to 25,000; Polysciences),Polyvinyl Alcohol (“PVA”—mol wt 124,000 to 186,000; 99% hydrolyzed;Aldrich Chemical Co., Milwaukee Wis., USA), Dichloromethane (“DCM” or“methylene chloride”; HPLC grade Fisher scientific), and distilledwater.

A. Preparation of 5% (w/v) Polymer Solutions

Depending on the polymer solution being prepared, 1.00 g of PCL or PLA,or 0.50 g each of PLA and washed EVA is weighed directly into a 20 mlglass scintillation vial. Twenty milliliters of DCM is then added, andthe vial tightly capped. The vial is stored at room temperature (25° C.)for one hour (occasional shaking may be used), or until all the polymerhas dissolved (the solution should be clear). The solution may be storedat room temperature for at least two weeks.

B. Preparation of 5% (w/v) Stock Solution of PVA

Twenty-five grams of PVA is weighed directly into a 600 ml glass beaker.Five hundred milliliters of distilled water is added, along with a 3inch Teflon coated stir bar. The beaker is covered with glass todecrease evaporation losses, and placed into a 2000 ml glass beakercontaining 300 ml of water (which acts as a water bath). The PVA isstirred at 300 rpm at 85° C. (Corning hot plate/stirrer) for 2 hours oruntil fully dissolved. Dissolution of the PVA may be determined by avisual check; the solution should be clear. The solution is thentransferred to a glass screw top storage container and stored at 4° C.for a maximum of two months. The solution, however should be warmed toroom temperature before use or dilution.

C. Procedure for Producing Microspheres

Based on the size of microspheres being made (see Table I), 100 ml ofthe PVA solution (concentrations given in Table I) is placed into the200 ml water jacketed beaker. Haake circulating water bath is connectedto this beaker and the contents are allowed to equilibrate at 27° C.(+/−1° C.) for 10 minutes. Based on the size of microspheres being made(see Table I), the start speed of the overhead stirrer is set, and theblade of the overhead stirrer placed half way down in the PVA solution.The stirrer is then started, and 10 ml of polymer solution (polymersolution used based on type of microspheres being produced) is thendripped into the stirring PVA over a period of 2 minutes using a 5 mlautomatic pipetter. After 3 minutes the stir speed is adjusted (seeTable I), and the solution stirred for an additional 2.5 hours. Thestirring blade is then removed from the microsphere preparation, andrinsed with 10 ml of distilled water so that the rinse solution drainsinto the microsphere preparation. The microsphere preparation is thenpoured into a 500 ml beaker, and the jacketed water bath washed with 70ml of distilled water, which is also allowed to drain into themicrosphere preparation. The 180 ml microsphere preparation is thenstirred with a glass rod, and equal amounts are poured into fourpolypropylene 50 ml centrifuge tubes. The tubes are then capped, andcentrifuged for 10 minutes (force given in Table I). A 5 ml automaticpipetter or vacuum suction is then utilized to draw 45 ml of the PVAsolution off of each microsphere pellet. TABLE I PVA concentrations,stir speeds, and centrifugal force requirements for each diameter rangeof microspheres. PRODUCTION MICROSPHERE DIAMETER RANGES STAGE 30 μm to100 μm 10 μm to 30 μm 0.1 μm to 3 μm PVA 2.5% (w/v) (i.e.,) 5% (w/v)(i.e., 3.5% (w/v) (i.e., concentration dilute 5% stock undiluted stock)dilute 5% stock with distilled water with distilled water Starting Stir500 rpm +/− 50 rpm 500 rpm +/− 50 rpm 3000 rpm +/− 200 rpm SpeedAdjusted Stir Speed 500 rpm +/− 50 rpm 500 rpm +/− 50 rpm 2500 rpm +/−200 rpm Centrifuge 1000 g +/− 100 g 1000 g +/− 100 g 10 000 g +/− 1000 gForce (Table top model) (Table top model) (High speed model)

Five milliliters of distilled water is then added to each centrifugetube, which is then vortexed to resuspend the microspheres. The fourmicrosphere suspensions are then pooled into one centrifuge tube alongwith 20 ml of distilled water, and centrifuged for another 10 minutes(force given in Table I). This process is repeated two additional timesfor a total of three washes. The microspheres are then centrifuged afinal time, and resuspended in 10 ml of distilled water. After the finalwash, the microsphere preparation is transferred into a preweighed glassscintillation vial. The vial is capped, and left overnight at roomtemperature (25° C.) in order to allow the microspheres to sediment outunder gravity. Microspheres which fall in the size range of 0.1 um to 3um do not sediment out under gravity, so they are left in the 10 mlsuspension.

D. Drying of 10 μm to 30 μm or 30 μm to 100 μm Diameter Microspheres

After the microspheres have sat at room temperature overnight, a 5 mlautomatic pipetter or vacuum suction is used to draw the supernatant offof the sedimented microspheres. The microspheres are allowed to dry inthe uncapped vial in a drawer for a period of one week or until they arefully dry (vial at constant weight). Faster drying may be accomplishedby leaving the uncapped vial under a slow stream of nitrogen gas (flowapprox. 10 ml/min.) in the fume hood. When fully dry (vial at constantweight), the vial is weighed and capped. The labeled, capped vial isstored at room temperature in a drawer. Microspheres are normally storedno longer than 3 months.

E. Drying of 0.1 μm to 3 μm Diameter Microspheres

This size range of microspheres will not sediment out, so they are leftin suspension at 4° C. for a maximum of four weeks. To determine theconcentration of microspheres in the 10 ml suspension, a 200 μl sampleof the suspension is pipetted into a 1.5 ml preweighed microfuge tube.The tube is then centrifuged at 10,000 g (Eppendorf table topmicrofuge), the supernatant removed, and the tube allowed to dry at 50°C. overnight. The tube is then reweighed in order to determine theweight of dried microspheres within the tube.

F. Manufacture of Paclitaxel Loaded Microsphere

In order to prepare paclitaxel containing microspheres, an appropriateamount of weighed paclitaxel (based upon the percentage of paclitaxel tobe encapsulated) is placed directly into a 20 ml glass scintillationvial. Ten milliliters of an appropriate polymer solution is then addedto the vial containing the paclitaxel, which is then vortexed until thepaclitaxel has dissolved.

Microspheres containing paclitaxel may then be produced essentially asdescribed above in steps (C) through (E).

Example 3 Surfactant Coated Microspheres

A. Materials and Methods

Microspheres were manufactured from Poly (DL) lactic acid (PLA), polymethylmethacrylate (PMMA), polycaprolactone (PCL) and 50:50 Ethylenevinyl acetate (EVA):PLA essentially as described in Example 2. Sizeranged from 10 to 100 um with a mean diameter 45 um.

Human blood was obtained from healthy volunteers. Neutrophils (whiteblood cells) were separated from the blood using dextran sedimentationand Ficoll Hypaque centrifugation techniques. Neutrophils were suspendedat 5 million cells per ml in Hanks Buffered Salt Solution (“HBSS”).

Neutrophil activation levels were determined by the generation ofreactive oxygen species as determined by chemiluminescence. Inparticular, chemiluminescence was determined by using an LKB luminometerwith 1 uM luminol enhancer. Plasma precoating (or opsonization) ofmicrospheres was performed by suspending 10 mg of microspheres in 0.5 mlof plasma and tumbling at 37° C. for 30 min.

Microspheres were then washed in 1 ml of HBSS and the centrifugedmicrosphere pellet added to the neutrophil suspension at 37° C. at timet=0. Microsphere surfaces were modified using a surfactant calledPluronic F127 (BASF) by suspending 10 mg of microspheres in 0.5 ml of 2%w/w solution of F127 in HBSS for 30 min at 37° C. Microspheres were thenwashed twice in 1 ml of HBSS before adding to neutrophils or to plasmafor further precoating.

B. Results

FIG. 1 shows that the untreated microspheres give chemiluminescencevalues less than 50 mV. These values represent low levels of neutrophilactivation. By way of comparison, inflammatory microcrystals might givevalues close to 1000 mV, soluble chemical activators might give valuesclose to 5000 mV. However, when the microspheres are precoated withplasma, all chemiluminescence values are amplified to the 100 to 300 mVrange (see FIG. 1). These levels of neutrophil response or activationcan be considered mildly inflammatory. PMMA gave the biggest responseand could be regarded as the most inflammatory. PLA and PCL both becomethree to four times more potent in activating neutrophils after plasmapretreatment (or opsonization) but there is little difference betweenthe two polymers in this regard. EVA:PLA is not likely to be used inangiogenesis formulations since the microspheres are difficult to dryand resuspend in aqueous buffer. This effect of plasma is termedopsonization and results from the adsorption of antibodies or complementmolecules onto the surface. These adsorbed species interact withreceptors on white blood cells and cause an amplified cell activation.

FIGS. 2-5 describe the effects of plasma precoating of PCL, PMMA, PLAand EVA:PLA respectively as well as showing the effect of pluronic F127precoating prior to plasma precoating of microspheres. These figures allshow the same effect: (1) plasma precoating amplifies the response; (2)Pluronic F127 precoating has no effect on its own; (3) the amplifiedneutrophil response caused by plasma precoating can be stronglyinhibited by pretreating the microsphere surface with 2% pluronic F127.

The nature of the adsorbed protein species from plasma was also studiedby electrophoresis. Using this method, it was shown that pretreating thepolymeric surface with Pluronic F127 inhibited the adsorption ofantibodies to the polymeric surface.

FIGS. 6-9 likewise show the effect of precoating PCL, PMMA, PLA orEVA:PLA microspheres (respectively) with either IgG (2 mg/ml) or 2%pluronic F127 then IgG (2 mg/ml). As can be seen from these figures, theamplified response caused by precoating microspheres with IgG can beinhibited by treatment with pluronic F127.

This result shows that by pretreating the polymeric surface of all fourtypes of microspheres with Pluronic F127, the “inflammatory” response ofneutrophils to microspheres may be inhibited.

Example 4 Encapsulation of Paclitaxel

Five hundred micrograms of either paclitaxel or baccatin (a paclitaxelanalog, available from Inflazyme Pharmaceuticals Inc., Vancouver,British Columbia, Canada) are dissolved in 1 ml of a 50:50ELVAX:poly-1-lactic acid mixture in dcm. Microspheres are then preparedin a dissolution machine (Six-spindle dissolution tester, VanderKanp,Van Kell Industries Inc., U.S.A.) in triplicate at 200 rpm, 42° C., for3 hours. Microspheres so prepared are washed twice in water and sized onthe microscope.

Determination of paclitaxel encapsulation is undertaken in a uv/visassay (uv/vis lambda max. at 237 nm, fluorescence assay at excitation237, emission at 325 nm; Fluorescence results are presented in squarebrackets [ ]). Utilizing the procedures described above, 58 μg (+/−12μg) [75 μg (+/−25 μg)] of paclitaxel may be encapsulated from a total500 μg of starting material. This represents 12% (+/−2.4%) [15% (+/−5%)]of the original weight, or 1.2% (+/−0.25%) [1.5% (+/−0.5%)] by weight ofthe polymer. After 18 hours of tumbling in an oven at 37° C., 10.3%(+/−10%) [6% (+/−5.6%)] of the total paclitaxel had been released fromthe microspheres.

For baccatin, 100+/−15 μg [83+/−23 μg] of baccatin can be encapsulatedfrom a total of 500 μg starting material. This represents a 20% (+/−3%)[17% (+/−5%) of the original weight of baccatin, and 2% (+/−0.3%) [1.7%(+/−0.5%)] by weight of the polymer. After 18 hours of tumbling in anoven at 37° C., 55% (+/−13%) [60% (+/−23%)] of the baccatin is releasedfrom the microspheres.

Example 5 Controlled Delivery of Paclitaxel from Microspheres Composedof a Blend of Ethylene-Vinyl-Acetate Copolymer and Poly (D,L LacticAcid). In Vivo Testing of the Microspheres on the Cam Assay

This example describes the preparation of paclitaxel-loaded microspherescomposed of a blend of biodegradable poly (d,l-lactic acid) (PLA)polymer and nondegradable ethylene-vinyl acetate (EVA) copolymer. Inaddition, the in vitro release rate and anti-angiogenic activity ofpaclitaxel released from microspheres placed on a CAM are demonstrated.

Reagents which were utilized in these experiments include paclitaxel,which is purchased from Sigma Chemical Co. (St. Louis, Mo.); PLA(molecular weight 15,000-25,000) and EVA (60% vinyl acetate) (purchasedfrom Polysciences (Warrington, Pa.); polyvinyl alcohol (PVA) (molecularweight 124,000-186,000, 99% hydrolysed, purchased from Aldrich ChemicalCo. (Milwaukee, Wis.)) and Dichloromethane (DCM) (HPLC grade, obtainedfrom Fisher Scientific Co). Distilled water is used throughout.

A. Preparation of Microspheres

Microspheres are prepared essentially as described in Example 2utilizing the solvent evaporation method. Briefly, 5% w/v polymersolutions in 20 mL DCM are prepared using blends of EVA:PLA between35:65 to 90:10. To 5 mL of 2.5% w/v PVA in water in a 20 mL glass vialis added 1 mL of the polymer solution dropwise with stirring. Sixsimilar vials are assembled in a six position overhead stirrer,dissolution testing apparatus (Vanderkamp) and stirred at 200 rpm. Thetemperature of the vials is increased from room temperature to 40° C.over 15 min and held at 40° C. for 2 hours. Vials are centrifuged at500×g and the microspheres washed three times in water. At some EVA:PLApolymer blends, the microsphere samples aggregated during the washingstage due to the removal of the dispersing or emulsifying agent, PVA.This aggregation effect could be analyzed semi-quantitatively sinceaggregated microspheres fused and the fused polymer mass floated on thesurface of the wash water. This surface polymer layer is discardedduring the wash treatments and the remaining, pelleted microspheres areweighed.The % aggregation is determined from${\%\quad{aggregation}} = \frac{1 - {\left( {{weight}\quad{of}\quad{pelleted}\quad{microspheres}} \right) \times 100}}{{initial}\quad{polymer}\quad{weight}}$

Paclitaxel loaded microspheres (0.6% w/w paclitaxel) are prepared bydissolving the paclitaxel in the 5% w/v polymer solution in DCM. Thepolymer blend used is 50:50 EVA:PLA. A “large” size fraction and “small”size fraction of microspheres are produced by adding thepaclitaxel/polymer solution dropwise into 2.5% w/v PVA and 5% w/v PVA,respectively. The dispersions are stirred at 40° C. at 200 rpm for 2hours, centrifuged and washed 3 times in water as described previously.Microspheres are air dried and samples are sized using an opticalmicroscope with a stage micrometer. Over 300 microspheres are countedper sample. Control microspheres (paclitaxel absent) are prepared andsized as described previously.

B. Encapsulation Efficiency

Known weights of paclitaxel-loaded microspheres are dissolved in 1 mLDCM, 20 mL of 40% acetonitrile in water at 50° C. are added and vortexeduntil the DCM had been evaporated. The concentration of paclitaxel inthe 40% acetonitrile is determined by HPLC using a mobile phase ofwater:methanol:acetonitrile (37:5:58) at a flow rate of 1 mL/min(Beckman isocratic pump), a C8 reverse phase column (Beckman) and UVdetection at 232 nm. To determine the recovery efficiency of thisextraction procedure, known weights of paclitaxel from 100-1000 μg aredissolved in 1 mL of DCM and subjected to the same extraction procedurein triplicate as described previously. Recoveries are always greaterthan 85% and the values of encapsulation efficiency are correctedappropriately.

C. Drug Release Studies

In 15 mL glass, screw capped tubes are placed 10 mL of 10 mM phosphatebuffered saline (PBS), pH 7.4 and 35 mg paclitaxel-loaded microspheres.The tubes are tumbled at 37° C. and at given time intervals, centrifugedat 1500×g for 5 min and the supernatant saved for analysis. Microspherepellets are resuspended in fresh PBS (10 mL) at 37° C. and reincubated.Paclitaxel concentrations are determined by extraction into 1 mL DCMfollowed by evaporation to dryness under a stream of nitrogen,reconstitution in 1 mL of 40% acetonitrile in water and analysis usingHPLC as previously described.

D. Scanning Electron Microscopy (SEM)

Microspheres are placed on sample holders, sputter coated with gold andmicrographs obtained using a Philips 501B SEM operating at 15 kV.

E. CAM Studies

Fertilized, domestic chick embryos are incubated for 4 days prior toshell-less culturing. The egg contents are incubated at 90% relativehumidity and 3% CO₂ for 2 days. On day 6 of incubation, 1 mg aliquots of0.6% paclitaxel loaded or control (paclitaxel free) microspheres areplaced directly on the CAM surface. After a 2 day exposure thevasculature is examined using a stereomicroscope interfaced with a videocamera; the video signals are then displayed on a computer and videoprinted.

F. Results

Microspheres prepared from 100% EVA are freely suspended in solutions ofPVA but aggregated and coalesced or fused extensively on subsequentwashing in water to remove the PVA. Blending EVA with an increasingproportion of PLA produced microspheres showing a decreased tendency toaggregate and coalesce when washed in water, as described in FIG. 10A. A50:50 blend of EVA:PLA formed microspheres with good physical stability,that is the microspheres remained discrete and well suspended withnegligible aggregation and coalescence.

The size range for the “small” size fraction microspheres is determinedto be >95% of the microsphere sample (by weight) between 10-30 mm andfor the “large” size fraction, >95% of the sample (by weight) between30-100 mm. Representative scanning electron micrographs of paclitaxelloaded 50:50 EVA:PLA microspheres in the “small” and “large” size rangesare shown in FIGS. 10B and 10C, respectively. The microspheres arespherical with a smooth surface and with no evidence of solid drug onthe surface of the microspheres. The efficiency of loading 50:50 EVA:PLAmicrospheres with paclitaxel is between 95-100% at initial paclitaxelconcentrations of between 100-1000 mg paclitaxel per 50 mg polymer.There is no significant difference (Student t-test, p<0.05) between theencapsulation efficiencies for either “small” or “large” microspheres.

The time course of paclitaxel release from 0.6% w/v loaded 50:50 EVA:PLAmicrospheres is shown in FIG. 10D for “small” size (open circles) and“large” size (closed circles) microspheres. The release rate studies arecarried out in triplicate tubes in 3 separate experiments. The releaseprofiles are biphasic with an initial rapid release of paclitaxel or“burst” phase occurring over the first 4 days from both size rangemicrospheres. This is followed by a phase of much slower release. Thereis no significant difference between the release rates from “small” or“large” microspheres. Between 10-13% of the total paclitaxel content ofthe microspheres is released in 50 days.

The paclitaxel loaded microspheres (0.6% w/v loading) are tested usingthe CAM assay and the results are shown in FIG. 10E. The paclitaxelmicrospheres released sufficient drug to produce a zone of avascularityin the surrounding tissue (FIG. 10F). Note that immediately adjacent tothe microspheres (“MS” in FIGS. 10E and 10F) is an area in which bloodvessels are completely absent (Zone 1); further from the microspheres isan area of disrupted, non-functioning capillaries (Zone 2); it is onlyat a distance of approximately 6 mm from the microspheres that thecapillaries return to normal. In CAMs treated with control microspheres(paclitaxel absent) there is a normal capillary network architecture(figure not shown.)

Discussion

Peritubular drug administration is a mildly invasive surgical technique.Therefore, ideally, a perivascular formulation of an anti-proliferativedrug such as paclitaxel would release the drug at the tumor or diseasesite at concentrations sufficient for activity for a prolonged period oftime, of the order of several months. EVA is a tissue compatiblenondegradable polymer which has been used extensively for the controlleddelivery of macromolecules over long time periods (>100 days).

EVA is initially selected as a polymeric, biomaterial for preparingmicrospheres with paclitaxel dispersed in the polymer matrix. However,microspheres prepared with 100% EVA aggregated and coalesced almostcompletely during the washing procedure.

Polymers and copolymers based on lactic acid and glycolic acid arephysiologically inert and biocompatible and degrade by hydrolysis totoxicologically acceptable products. Copolymers of lactic acid andglycolic acids have faster degradation rates than PLA and drug loadedmicrospheres prepared using these copolymers are unsuitable forprolonged, controlled release over several months.

FIG. 10A shows that increasing the proportion of PLA in a EVA:PLA blenddecreased the extent of aggregation of the microsphere suspensions.Blends of 50% or less EVA in the EVA:PLA matrix produced physicallystable microsphere suspensions in water or PBS. A blend of 50:50 EVA:PLAis selected for all subsequent studies.

Different size range fractions of microspheres could be prepared bychanging the concentration of the emulsifier, PVA, in the aqueous phase.“Small” microspheres are produced at the higher PVA concentration of 5%w/v whereas “large” microspheres are produced at 2.5% w/v PVA. All otherproduction variables are the same for both microsphere size fractions.The higher concentration of emulsifier gave a more viscous aqueousdispersion medium and produced smaller droplets ofpolymer/paclitaxel/DCM emulsified in the aqueous phase and thus smallermicrospheres. The paclitaxel loaded microspheres contained between95-100% of the initial paclitaxel added to the organic phaseencapsulated within the solid microspheres. The low water solubility ofpaclitaxel favoured partitioning into the organic phase containing thepolymer.

Release rates of paclitaxel from the 50:50 EVA:PLA microspheres are veryslow with less than 15% of the loaded paclitaxel being released in 50days. The initial burst phase of drug release may be due to diffusion ofdrug from the superficial region of the microspheres (close to themicrosphere surface).

The mechanism of drug release from nondegradable polymeric matrices suchas EVA is thought to involve the diffusion of water through thedispersed drug phase within the polymer, dissolution of the drug anddiffusion of solute through a series of interconnecting, fluid filledpores. Blends of EVA and PLA have been shown to be immiscible orbicontinuous over a range of 30 to 70% EVA in PLA. In degradationstudies in PBS buffer at 37° C., following an induction or lag period,PLA hydrolytically degraded and eroded from the EVA:PLA polymer blendmatrix leaving an inactive sponge-like skeleton. Although the inductionperiod and rate of PLA degradation and erosion from the blended matricesdepended on the proportion of PLA in the matrix and on process history,there is consistently little or no loss of PLA until after 40-50 days.

Although some erosion of PLA from the 50:50 EVA:PLA microspheres mayhave occurred within the 50 days of the in vitro release rate study(FIG. 10C), it is likely that the primary mechanism of drug release fromthe polymer blend is diffusion of solute through a pore network in thepolymer matrix.

At the conclusion of the release rate study, the microspheres areanalyzed from the amount of drug remaining. The values for the percentof paclitaxel remaining in the 50 day incubation microsphere samples are94%+/−9% and 89%+/−12% for “large” and “small” size fractionmicrospheres, respectively.

Microspheres loaded with 6 mg per mg of polymer (0.6%) providedextensive inhibition of angiogenesis when placed on the CAM of theembryonic chick (FIGS. 10E and 10F).

Example 6 Therapeutic Agent Encapsulation in Poly(ε-Caprolactone)Microspheres. Inhibition of Angiogenesis on the Cam Assay byPaclitaxel-Loaded Microspheres

This example evaluates the in vitro release rate profile of paclitaxelfrom biodegradable microspheres of poly(ε-caprolactone) and demonstratesthe in vivo anti-angiogenic activity of paclitaxel released from thesemicrospheres when placed on the CAM.

Reagents which were utilized in these experiments include:poly(ε-caprolactone) (“PCL”) (molecular weight 35,000-45,000; purchasedfrom Polysciences (Warrington, Pa.)); dichloromethane (“DCM”) fromFisher Scientific Co., Canada; polyvinyl alcohol (PVP) (molecular weight12,000-18,000, 99% hydrolysed) from Aldrich Chemical Co. (Milwaukee,Wis.), and paclitaxel from Sigma Chemical Co. (St. Louis, Mo.). Unlessotherwise stated all chemicals and reagents are used as supplied.Distilled water is used throughout.

A. Preparation of Microspheres

Microspheres are prepared essentially as described in Example 2utilizing the solvent evaporation method. Briefly, 5% w/w paclitaxelloaded microspheres are prepared by dissolving 10 mg of paclitaxel and190 mg of PCL in 2 ml of DCM, adding to 100 ml of 1% PVP aqueoussolution and stirring at 1000 rpm at 25° C. for 2 hours. The suspensionof microspheres is centrifuged at 1000×g for 10 minutes (Beckman GPR),the supernatant removed and the microspheres washed three times withwater. The washed microspheres are air-dried overnight and stored atroom temperature. Control microspheres (paclitaxel absent) are preparedas described above. Microspheres containing 1% and 2% paclitaxel arealso prepared. Microspheres are sized using an optical microscope with astage micrometer.

B. Encapsulation Efficiency

A known weight of drug-loaded microspheres (about 5 mg) is dissolved in8 ml of acetonitrile and 2 ml distilled water is added to precipitatethe polymer. The mixture is centrifuged at 1000 g for 10 minutes and theamount of paclitaxel encapsulated is calculated from the absorbance ofthe supernatant measured in a UV spectrophotometer (Hewlett-Packard8452A Diode Array Spectrophotometer) at 232 nm.

C. Drug Release Studies

About 10 mg of paclitaxel-loaded microspheres are suspended in 20 ml of10 mM phosphate buffered saline, pH 7.4 (PBS) in screw-capped tubes. Thetubes are tumbled end-over-end at 37° C. and at given time intervals19.5 ml of supernatant is removed (after allowing the microspheres tosettle at the bottom), filtered through a 0.45 um membrane filter andretained for paclitaxel analysis. An equal volume of PBS is replaced ineach tube to maintain sink conditions throughout the study. Thefiltrates are extracted with 3×1 ml DCM, the DCM extracts evaporated todryness under a stream of nitrogen, redissolved in 1 ml acetonitrile andanalyzed by HPLC using a mobile phase of water:methanol:acetonitrile(37:5:58) at a flow rate of 1 ml min⁻¹ (Beckman Isocratic Pump), a C8reverse phase column (Beckman), and UV detection (Shimadzu SPD A) at 232μm.

D. CAM Studies

Fertilized, domestic chick embryos are incubated for 4 days prior toshell-less culturing. On day 6 of incubation, 1 mg aliquots of 5%paclitaxel-loaded or control (paclitaxel-free) microspheres are placeddirectly on the CAM surface. After a 2-day exposure the vasculature isexamined using a stereomicroscope interfaced with a video camera; thevideo signals are then displayed on a computer and video printed.

E. Scanning Electron Microscopy

Microspheres are placed on sample holders, sputter-coated with gold andthen placed in a Philips 501B Scanning Electron Microscope operating at15 kV.

F. Results

The size range for the microsphere samples is between 30-100 um,although there is evidence in all paclitaxel-loaded or controlmicrosphere batches of some microspheres falling outside this range. Theefficiency of loading PCL microspheres with paclitaxel is always greaterthan 95% for all drug loadings studied. Scanning electron microscopydemonstrated that the microspheres are all spherical and many showed arough or pitted surface morphology. There appeared to be no evidence ofsolid drug on the surface of the microspheres.

The time courses of paclitaxel release from 1%, 2% and 5% loaded PCLmicrospheres are shown in FIG. 1I A. The release rate profiles arebi-phasic. There is an initial rapid release of paclitaxel or “burstphase” at all drug loadings. The burst phase occurred over 1-2 days at1% and 2% paclitaxel loading and over 3-4 days for 5% loadedmicrospheres. The initial phase of rapid release is followed by a phaseof significantly slower drug release. For microspheres containing 1% or2% paclitaxel there is no further drug release after 21 days. At 5%paclitaxel loading, the microspheres had released about 20% of the totaldrug content after 21 days.

FIG. 11B shows CAMs treated with control PCL microspheres, and FIG. 11Cshows treatment with 5% paclitaxel loaded microspheres. The CAM with thecontrol microspheres shows a normal capillary network architecture. TheCAM treated with paclitaxel-PCL microspheres shows marked vascularregression and zones which are devoid of a capillary network.

G. Discussion

The solvent evaporation method of manufacturing paclitaxel-loadedmicrospheres produced very high paclitaxel encapsulation efficiencies ofbetween 95-100%. This is due to the poor water solubility of paclitaxeland its hydrophobic nature favouring partitioning in the organic solventphase containing the polymer.

The biphasic release profile for paclitaxel is typical of the releasepattern for many drugs from biodegradable polymer matrices.Poly(ε-caprolactone) is an aliphatic polyester which can be degraded byhydrolysis under physiological conditions and it is non-toxic and tissuecompatible. The degradation of PCL is significantly slower than that ofthe extensively investigated polymers and copolymers of lactic andglycolic acids and is therefore suitable for the design of long-termperitubular drug delivery systems. The initial rapid or burst phase ofpaclitaxel release is thought to be due to diffusional release of thedrug from the superficial region of the microspheres (close to themicrosphere surface). Release of paclitaxel in the second (slower) phaseof the release profiles is not likely due to degradation or erosion ofPCL because studies have shown that under in vitro conditions in waterthere is no significant weight loss or surface erosion of PCL over a7.5-week period. The slower phase of paclitaxel release is probably dueto dissolution of the drug within fluid-filled pores in the polymermatrix and diffusion through the pores. The greater release rate athigher paclitaxel loading is probably a result of a more extensive porenetwork within the polymer matrix.

Paclitaxel microspheres with 5% loading have been shown to releasesufficient drug to produce extensive inhibition of angiogenesis whenplaced on the CAM. The inhibition of blood vessel growth resulted in anavascular zone as shown in FIG. 11C.

Example 7 Therapeutic Agent-Loaded Peritubular Polymeric Films Composedof Ethylene Vinyl Acetate and a Surfactant

Two types of films are investigated within this example: pure EVA filmsloaded with paclitaxel and EVA/surfactant blend films loaded withpaclitaxel.

The surfactants being examined are two hydrophobic surfactants (Span 80and Pluronic L101) and one hydrophilic surfactant (Pluronic F127). Thepluronic surfactants are themselves polymers, which is an attractiveproperty since they can be blended with EVA to optimize various drugdelivery properties. Span 80 is a smaller molecule which is in somemanner dispersed in the polymer matrix, and does not form a blend.

Surfactants is useful in modulating the release rates of paclitaxel fromfilms and optimizing certain physical parameters of the films. Oneaspect of the surfactant blend films which indicates that drug releaserates can be controlled is the ability to vary the rate and extent towhich the compound will swell in water. Diffusion of water into apolymer-drug matrix is critical to the release of drug from the carrier.FIGS. 12C and 12D show the degree of swelling of the films as the levelof surfactant in the blend is altered. Pure EVA films do not swell toany significant extent in over 2 months. However, by increasing thelevel of surfactant added to the EVA it is possible to increase thedegree of swelling of the compound, and by increasing hydrophilicityswelling can also be increased.

Results of experiments with these films are shown below in FIGS. 12A-E.Briefly, FIG. 12A shows paclitaxel release (in mg) over time from pureEVA films. FIG. 12B shows the percentage of drug remaining for the samefilms. As can be seen from these two figures, as paclitaxel loadingincreases (i.e., percentage of paclitaxel by weight is increased), drugrelease rates increase, showing the expected concentration dependence.As paclitaxel loading is increased, the percent paclitaxel remaining inthe film also increases, indicating that higher loading may be moreattractive for long-term release formulations.

Physical strength and elasticity of the films is assessed in FIG. 12E.Briefly, FIG. 12E shows stress/strain curves for pure EVA andEVA-Surfactant blend films. This crude measurement of stressdemonstrates that the elasticity of films is increased with the additionof Pluronic F127, and that the tensile strength (stress on breaking) isincreased in a concentration dependent manner with the addition ofPluronic F127. Elasticity and strength are important considerations indesigning a film which can be manipulated for particular peritubularclinical applications without causing permanent deformation of thecompound.

The above data demonstrates the ability of certain surfactant additivesto control drug release rates and to alter the physical characteristicsof the vehicle.

Example 8 Incorporating Methoxypolyethylene Glycol 350 (MePEG) intoPoly(E-Caprolactone) to Develop a Formulation for the ControlledDelivery of Therapeutic Agents from a Paste

Reagents and equipment which were utilized within these experimentsinclude methoxypolyethylene glycol 350 (“MePEG”—Union Carbide, Danbury,Conn.). MePEG is liquid at room temperature, and has a freezing point of10° C. to −5° C.

A. Preparation of a MePEG/PCL Paclitaxel-Containing Paste

MePEG/PCL paste is prepared by first dissolving a quantity of paclitaxelinto MePEG, and then incorporating this into melted PCL. One advantagewith this method is that no DCM is required.

B. Analysis of Melting Point

The melting point of PCL/MePEG polymer blends may be determined bydifferential scanning calorimetry from 30° C. to 70° C. at a heatingrate of 2.5° C. per minute. Results of this experiment are shown inFIGS. 13A and 13B. Briefly, as shown in FIG. 13A the melting point ofthe polymer blend (as determined by thermal analysis) is decreased byMePEG in a concentration dependent manner. The melting point of thepolymer blends as a function of MePEG concentration is shown in FIG.13A. This lower melting point also translates into an increased time forthe polymer blends to solidify from melt as shown in FIG. 13B. A 30:70blend of MePEG:PCL takes more than twice as long to solidify from thefluid melt than does PCL alone.

C. Measurement of Brittleness

Incorporation of MePEG into PCL appears to produce a less brittle solid,as compared to PCL alone. As a “rough” way of quantitating this, aweighted needle is dropped from an equal height into polymer blendscontaining from 0% to 30% MePEG in PCL, and the distance that the needlepenetrates into the solid is then measured. The resulting graph is shownas FIG. 13C. Points are given as the average of four measurements +/−1S.D.

For purposes of comparison, a sample of paraffin wax is also tested andthe needle penetrated into this a distance of 7.25 mm+/−0.3 mm.

D. Measurement of Paclitaxel Release

Pellets of polymer (PCL containing 0%, 5%, 10% or 20% MePEG) areincubated in phosphate buffered saline (PBS, pH 7.4) at 37° C., and %change in polymer weight is measured over time. As can be seen in FIG.13D, the amount of weight lost increases with the concentration of MePEGoriginally present in the blend. It is likely that this weight loss isdue to the release of MePEG from the polymer matrix into the incubatingfluid. This would indicate that paclitaxel will readily be released froma MePEG/PCL blend since paclitaxel is first dissolved in MePEG beforeincorporation into PCL.

E. Effect of Varying Quantities of MePEG on Paclitaxel Release

Thermopastes are made up containing between 0.8% and 20% MePEG in PCL.These are loaded with 1% paclitaxel. The release of paclitaxel over timefrom 10 mg pellets in PBS buffer at 37° C. is monitored using HPLC. Asis shown in FIG. 13E, the amount of MePEG in the formulation does notaffect the amount of paclitaxel that is released.

F. Effect of Varying Quantities of Paclitaxel on the Total Amount ofPaclitaxel Released From a 20% MePEG/PCL Blend

Thermopastes are made up containing 20% MePEG in PCL and loaded withbetween 0.2% and 10% paclitaxel. The release of paclitaxel over time ismeasured as described above. As shown in FIG. 13F, the amount ofpaclitaxel released over time increases with increased paclitaxelloading. When plotted as the percent total paclitaxel released, however,the order is reversed (FIG. 13G). This gives information about theresidual paclitaxel remaining in the paste and allows for a projectionof the period of time over which paclitaxel may be released from the 20%MePEG Thermopaste.

G. Strength Analysis of Various MePEG/PCL Blends

A CT-40 mechanical strength tester is used to measure the strength ofsolid polymer “tablets” of diameter 0.88 cm and an average thickness of0.560 cm. The polymer tablets are blends of MePEG at concentrations of0%, 5%, 10% or 20% in PCL.

Results of this test are shown in FIG. 13H, where both the tensilestrength and the time to failure are plotted as a function of % MePEG inthe blend. Single variable ANOVA indicated that the tablet thicknesseswithin each group are not different. As can be seen from FIG. 13H, theaddition of MePEG into PCL decreased the hardness of the resultingsolid.

Example 9 Alteration of Therapeutic Agent Release from Thermopaste usingLow Molecular Weight Poly(D,L, Lactic Acid)

As discussed above, depending on the desired therapeutic effect, eitherquick release or slow release polymeric carriers may be desired. Forexample, polycaprolactone (PCL) and mixtures of PCL with poly(ethyleneglycol) (PEG) produce compositions which release paclitaxel over aperiod of several months. In particular, the diffusion of paclitaxel inthe polymers is very slow due to its large molecular size and extremehydrophobicity.

On the other hand, low molecular weight poly(DL-lactic acid) (PDLLA)gives fast degradation, ranging from one day to a few months dependingon its initial molecular weight. The release of paclitaxel, in thiscase, is dominated by polymer degradation. Another feature of lowmolecular weight PDLLA is its low melting temperature, (i.e., 40° C.-60°C.), which makes it suitable material for making Thermopaste. Asdescribed in more detail below, several different methods can beutilized in order to control the polymer degradation rate, including,for example, by changing molecular weight of the PDLLA, and/or by mixingit with high mol wt. PCL, PDLLA, or poly(lactide-co-glyocide) (PLGA).

A. Experimental Materials

D,L-lactic acid was purchased from Sigma Chemical Co., St. Louis, Mo.PCL (molecular weight 10-20,000) was obtained from Polysciences,Warrington, Pa. High molecular weight PDLLA (intrinsic viscosity 0.60dl/g) and PLGA (50:50 composition, viscosity 0.58 dl/g) were fromBirmingham Polymers.

B. Synthesis of Low Molecular Weight PDLLA

Low molecular weight PDLLA was synthesized from DL-lactic acid throughpolycondensation. Briefly, DL-lactic acid was heated in a glass beakerat 200° C. with nitrogen purge and magnetic stirring for a desired time.The viscosity increased during the polymerization, due to the increaseof molecular weight. Three batches were obtained with differentpolymerization times, i.e., 40 min (molecular weight 800), 120 min, 160min.

C. Formulation of Paclitaxel Thermopastes

Paclitaxel was loaded, at 20%, into the following materials by handmixing at a temperature about 60° C.

1. low molecular weight PDLLA with polymerization time of 40 min.

2. low molecular weight PDLLA with polymerization time of 120 min.

3. low mol. wt PDLLA with polymerization time of 160 min.

4. a mixture of 50:50 high molecular weight PDLLA and low molecularweight PDLLA 40 min.

5. a mixture of 50:50 high molecular weight PLGA and low molecularweight PDLLA 40 min.

6. mixtures of high molecular weight PCL and low molecular weight. PDLLA40 min with PCL:PDLLA of 10:90, 20:80, 40:60, 60:40, and 20:80.

Mixtures of high molecular weight PDLLA or PLGA with low molecularweight. PDLLA were obtained by dissolving the materials in acetonefollowed by drying.

D. Release Study

The release of paclitaxel into PBS albumin buffer at 37° C. was measuredas described above with HPLC at various times.

E. Results

Low molecular weight PDLLA 40 min was a soft material with light yellowcolor. The color is perhaps due to the oxidation during thepolycondensation. Low molecular weight PDLLA 120 min (yellow) and 160min (brown) were brittle solids at room temperature. They all becomemelts at 60° C. Mixtures of 50:50 high molecular weight PDLLA or PLGAwith low molecular weight PDLLA 40 min also melted about 60° C.

During the release, low molecular weight PDLLA 40 min and 120 min brokeup into fragments within one day, other materials were intact up to thiswriting (3 days).

The release of paclitaxel from formulations 2-5 were shown in FIG. 14.Low molecular weight PDLLA 40 min and 120 min gave the fastest releasedue to the break up of the paste. The release was perhaps solubilitylimited. Low molecular weight PDLLA 160 min. also gave a fast releaseyet maintained an intact pellet. For example, 10% of loaded paclitaxelwas released with one day. The 50:50 mixtures of high molecular weightPDLLA or PLGA with low molecular weight PDLLA 40 min were slower, i.e.,3.4% and 2.2% release within one day.

Although not specifically set forth above, a wide variety of otherpolymeric carriers may be manufactured, including for example, (1) lowmolecular weight (500-10,000) poly(D,L-lactic acid), poly(L-lacticacid), poly(glycolic acid), poly(6-hydroxycaproic acid),poly(5-hydroxyvaleric acid), poly(4-hydroxybutyric acid), and theircopolymers; (2) blends of above (#1) above; (3) blends of (#1) abovewith high molecular weight poly(DL-lactic acid), poly(L-lactic acid),poly(glycolic acid), poly(6-hydroxycaproic acid), poly(5-hydroxyvalericacid), poly(4-hydroxybutyric acid), and their copolymers; and (4)copolymers of poly(ethylene glycol) and pluronics with poly(D,L-lacticacid), poly(L-lactic acid), poly(glycolic acid), poly(6-hydroxycaproicacid), poly(5-hydroxyvaleric acid), poly(4-hydroxybutyric acid), andtheir copolymers.

Example 10 Preparation of Polymeric Compositions Containing WaterSoluble Additives and Paclitaxel

A. Preparation of Polymeric Compositions

Microparticles of co-precipitates of paclitaxel/additive were preparedand subsequently added to PCL to form pastes. Briefly, paclitaxel (100mg) was dissolved in 0.5 ml of ethanol (95%) and mixed with the additive(100 mg) previously dissolved or dispersed in 1.0 ml of distilled water.The mixture was triturated until a smooth paste was formed. The pastewas spread on a Petri dish and air-dried overnight at 37° C. The driedmass was pulverized using a mortar and pestle and passed through a mesh#140 (106 μm) sieve (Endecotts Test Sieves Ltd, London, England). Themicroparticles (40%) were then incorporated into molten PCL (60%) at 65°C. corresponding to a 20% loading of paclitaxel. The additives used inthe study were gelatin (Type B, 100 bloom, Fisher Scientific),methylcellulose, (British Drug Houses), dextran, T500 (Pharmacia,Sweden), albumin (Fisher Scientific), and sodium chloride (FisherScientific). Microparticles of paclitaxel and gelatin or albumin wereprepared as described above but were passed through a mesh # 60 (270 μm)sieve (Endecotts Test Sieves Ltd, London, England) to evaluate theeffect of microparticle size on the release of paclitaxel from thepaste. Pastes were also prepared to contain 10, 20 or 30% gelatin and20% paclitaxel in PCL to study the effect of the proportion of theadditive on drug release. Unless otherwise specified, pastes containing20% paclitaxel dispersed in PCL were prepared to serve as controls forthe release rate studies.

B. Drug Release Studies

Approximately 2.5 mg pellet of paclitaxel-loaded paste was suspended in50 ml of 10 mM phosphate buffered saline, pH 7.4 (PBS) in screw-cappedtubes. The tubes were tumbled end-over-end at 37° C. and at given timeintervals 49.5 ml of supernatant was removed, filtered through a 0.45 μmmembrane filter and retained for paclitaxel analysis. An equal volume ofPBS was replaced in each tube to maintain sink conditions throughout thestudy. For analysis, the filtrates were extracted with 3×1 mldichloromethane (DCM), the DCM extracts evaporated to dryness under astream of nitrogen and redissolved in 1 ml acetonitrile. The analysiswas by HPLC using a mobile phase of water:methanol:acetonitrile(37:5:58:) at a flow rate of 1 ml min⁻ (Beckman Isocratic Pump), a C18reverse phase column (Beckman), and UV detection (Shimadzu SPD A) at 232nm.

C. Swelling Studies

Paclitaxel/additive/PCL pastes, prepared using paclitaxel-additivemicroparticles of mesh size # 140 (and #60 for gelatin only), wereextruded to form cylinders, pieces were cut, weighed and the diameterand length of each piece were measured using a micrometer (MitutoyoDigimatic). The pieces were suspended in distilled water (10 ml) at 37°C. and at predetermined intervals the water was discarded and thediameter and the length of the cylindrical pieces were measured and thesamples weighed. The morphology of the samples (before and aftersuspending in water) was examined using scanning electron microscopy(SEM) (Hitachi F-2300). The samples were coated with 60% Au and 40% Pd(thickness 10-15 nm) using a Hummer Instrument (Technics, USA).

D. Chick Embryo Chorioallantoic Membrane (CAM) Studies

Fertilized, domestic chick embryos were incubated for 4 days prior toshell-less culturing. The egg contents were incubated at 90% relativehumidity and 3% CO₂ and on day 6 of incubation, 1 mg pieces of thepaclitaxel-loaded paste containing 6% paclitaxel, 24% gelatin and 70%PCL) or control (30% gelatin in PCL) pastes were placed directly on theCAM surface. After a 2-day exposure the vasculature was examined using astereomicroscope interfaced with a video camera; the video signals werethen displayed on a computer and video printed.

E. In Vivo Anti-Tumor Activity

Pastes, prepared as described above (using mesh size 140 fractions ofthe paclitaxel-gelatin microparticles) containing 20% paclitaxel, 20%gelatin and 60% PCL were filled into 8×1 ml syringes (BD InsulinSyringe, ½ cc) each syringe containing 150 mg of the paste (equivalentto 30 mg of paclitaxel). Ten week old DBA/2j female mice (16) weighing18-20 g were acclimatized for 4 days after arrival and each mouse wasinjected in the posteriolateral flank with MDAY-D2 tumor cells, (10×10⁶ml⁻¹) in 100 μl of phosphate buffered saline on day 1. On day 6, themice were divided into two groups of eight, the tumor site opened underanesthesia and 150 mg of the paste, previously heated to about 60° C.was extruded at the tumor site and the wound closed. One group wasimplanted with the paclitaxel-loaded paste and the other group withcontrol paste containing gelatin and PCL only. On day 16, the mice weresacrificed and the weight of the mice and the excised tumor weremeasured.

F. Results and Discussion

Microparticles of co-precipitated paclitaxel and gelatin or albumin werehard and brittle and were readily incorporated into PCL while the otheradditives produced soft particles which showed a tendency to break upduring the preparation of the paste.

FIG. 15 shows the time courses of paclitaxel release from pastescontaining 20% paclitaxel in PCL or 20% paclitaxel, 20% additive and 60%PCL. The release of paclitaxel from PCL with or without additivesfollowed a bi-phasic release pattern; initially, there was a faster drugrelease rate followed by a slower drug release of the drug. The initialperiod of faster release rate of paclitaxel from the pastes was thoughtto be due to dissolution of paclitaxel located on the surface ordiffusion of paclitaxel from the superficial regions of the paste. Thesubsequent slower phase of the release profiles may be attributed to adecrease in the effective surface area of the drug particles in contactwith the solvent, a slow ingress of the solvent into the polymer matrixor an increase in the mean diffusion paths of the drug through thepolymer matrix.

Both phases of the release profiles of paclitaxel from PCL increased inthe presence of the hydrophilic additives with gelatin, albumin andmethylcellulose producing the greatest increase in drug release rates(FIG. 15). There were further increases in the release of paclitaxelfrom the polymer matrix when larger paclitaxel-additive particles (270μm) were used to prepare the paste compared with the smallerpaclitaxel-additive particles (106 μm) were used (FIG. 16). Increases inthe amount of the additive (e.g., gelatin) produced a correspondingincrease in drug release (FIG. 16). FIG. 17A shows the swelling behaviorof pastes containing 20% paclitaxel, 20% additive and 60% PCL. The rateof swelling followed the ordergelatin>albumin>methylcellulose>dextran>sodium chloride. In addition,the rate of swelling increased when a higher proportion of thewater-soluble polymer was added to the paste (FIG. 17B). The pastescontaining gelatin or albumin swelled rapidly within the first 8-10hours and subsequently the rate of swelling decreased when the change inthe volume of the sample was greater than 40%. The paste prepared usingthe larger (270 μm) paclitaxel-gelatin particles swelled at a fasterrate than those prepared with the smaller (106 μm) paclitaxel-gelatinparticles. All pastes disintegrated when the volume increases weregreater than 50%. The SEM studies showed that the swelling of the pasteswas accompanied by the cracking of the matrix (FIG. 18). At highmagnifications (FIGS. 18C and 18D) there was evidence of needle or rodshaped paclitaxel crystals on the surface of the paste and in closeassociation with gelatin following swelling (FIGS. 18C and 18D).

Osmotic or swellable, hydrophilic agents embedded as discrete particlesin the hydrophobic polymer result in drug release by a combination ofthe erosion of the matrix, diffusion of drug through the polymer matrix,and/or diffusion and/or convective flow through pores created in thematrix by the dissolution of the water soluble additives. Osmotic agentsand swellable polymers dispersed in a hydrophobic polymer would imbibewater (acting as wicking agents), dissolve or swell and exert a turgorpressure which could rupture the septa (the polymer layer) betweenadjacent particles, creating microchannels and thus facilitate theescape of the drug molecules into the surrounding media by diffusion orconvective flow. The swelling and cracking of the paste matrix (FIG. 18)likely resulted in the formation of microchannels throughout theinterior of the matrix. The different rates and extent of swelling ofthe polymers (FIG. 17) may account for the differences in the observedpaclitaxel release rates (FIGS. 15 and 16).

FIG. 19 shows CAMs treated with control gelatin-PCL paste (FIG. 19A) and20% paclitaxel-gelatin-PCL paste (FIG. 19B). The paste on the surface ofthe CAMs are shown by the arrows in the figures. The CAM with thecontrol paste shows a normal capillary network architecture. The CAMstreated with paclitaxel-PCL paste consistently showed vascularregression and zones which were devoid of a capillary network.Incorporation of additives in the paste markedly increased the diameterof the zone of avascularity (FIG. 19).

The results of the in vivo study are shown in FIG. 20. Briefly,peri-tumoral injection of paclitaxel-gelatin-PCL paste into mice withestablished and palpable tumors showed that this preparation produced amean reduction of 63% in tumor mass compared with controls. In addition,there was no significant effect on the weights of the mice followingtreatment. Paclitaxel-PCL pastes (without additives) did not produce anysignificant reduction in tumor mass.

This study showed that the in vitro release of paclitaxel from PCL couldbe increased by the incorporation of paclitaxel/hydrophilic polymermicroparticles into PCL matrix. In vivo studies evaluating the efficacyof the formulation in treating subcutaneous tumors in mice also showedthat the paclitaxel/gelatin/PCL paste significantly reduced the tumormass. Factors such as the type of water soluble agent, the microparticlesize and the proportion of the additives were shown to influence therelease characteristics of the drug.

Peritubular injection of a chemotherapeutic paste into the adventitia ofa tube obstructed by malignant overgrowth can reduce local tumor growthand could relieve symptoms of obstruction without invasive surgicalprocedures.

Example 11 Modification of Paclitaxel Release from Thermopaste usingPDLLA-PEG-PDLLA and Low Molecular Weight Poly(D,L, Lactic Acid)

A. Preparation of PDLLA-PEG-PDLLA and Low Molecular Weight PDLLA

DL-lactide was purchased from Aldrich. Polyethylene glycol (PEG) withmolecular weight 8,000, stannous octoate, and DL-lactic acid wereobtained from Sigma. Poly-ε-caprolactone (PCL) with molecular weight20,000 was obtained from Birmingham Polymers (Birmingham, Ala.).Paclitaxel was purchased from Hauser Chemicals (Boulder, Colo.).Polystyrene standards with narrow molecular weight distributions werepurchased from Polysciences (Warrington, Pa.). Acetonitrile andmethylene chloride were HPLC grade (Fisher Scientific).

The triblock copolymer of PDLLA-PEG-PDLLA was synthesized by a ringopening polymerization. Monomers of DL-lactide and PEG in differentratios were mixed and 0.5 wt % stannous octoate was added. Thepolymerization was carried out at 150° C. for 3.5 hours. Low molecularweight PDLLA was synthesized through polycondensation of DL-lactic acid.The reaction was performed in a glass flask under the conditions ofgentle nitrogen purge, mechanical stirring, and heating at 180° C. for1.5 hours. The PDLLA molecular weight was about 800 measured bytitrating the carboxylic acid end groups.

B. Manufacture of Paste Formulations

Paclitaxel at loadings of 20% or 30% was thoroughly mixed into eitherthe PDLLA-PEG-PDLLA copolymers or blends of PDLLA:PCL 90:10, 80:20 and70:30 melted at about 60° C. The paclitaxel loaded pastes were weighedinto 1 ml syringes and stored at 4° C.

C. Characterization of PDLLA-PEG-PDLLA and the Paste Blends

The molecular weights and distributions of the PDLLA-PEG-PDLLAcopolymers were determined at ambient temperature by GPC using aShimadzu LC-10AD HPLC pump and a Shimadzu RID-6A refractive indexdetector (Kyoto, Japan) coupled to a 10⁴ Å Hewlett Packard Plgel column.The mobile phase was chloroform with a flow rate of 1 ml/min. Theinjection volume of the sample was 20 μl at a polymer concentration of0.2% (w/v). The molecular weights of the polymers were determinedrelative to polystyrene standards. The intrinsic viscosity ofPDLLA-PEG-PDLLA in CHCl₃ at 25° C. was measured with a Cannon-Fenskeviscometer.

Thermal analysis of the copolymers was carried out by differentialscanning calorimetry (DSC) using a TA Instruments 2000 controller andDuPont 910S DSC (Newcastle, Del.). The heating rate was 10° C./min andthe copolymer and paclitaxel/copolymer matrix samples were weighed (3-5mg) into crimped open aluminum sample pans.

H Nuclear magnetic resonance (NMR) was used to determine the chemicalcomposition of the polymer. ¹H NMR spectra of paclitaxel loadedPDLLA-PEG-PDLLA were obtained in CDCl₃ using an NMR instrument (Bruker,AC-200E) at 200 MHz. The concentration of the polymer was 1-2%.

The morphology of the paclitaxel/PDLLA-PEG-PDLLA paste was investigatedusing scanning electron microscopy (SEM) (Hitachi F-2300). The samplewas coated with 60% Au and 40% Pd (thickness 10-15 nm) using a Hummerinstrument (Technics, USA).

D. In Vitro Release of Paclitaxel

A small pellet of 20% paclitaxel loaded PDLLA:PCL paste (about 2 mg) ora cylinder (made by extrusion melten paste through a syringe withoutneedle) of 20% paclitaxel loaded PDLLA-PEG-PDLLA paste were put intocapped 14 ml glass tubes containing 10 ml phosphate buffered saline(PBS, pH 7.4) with 0.4 g/L albumin. The tube was incubated at 37° C.with gentle rotational mixing. The supernatant was withdrawnperiodically for paclitaxel analysis and replaced with fresh PBS/albuminbuffer. The supernatant (10 ml) was extracted with 1 ml methylenechloride. The water phase was decanted and the methylene chloride phasewas dried under a stream of nitrogen at 60° C. The dried residue wasreconstituted in a 40:60 water:acetonitrile mixture and centrifuged at10,000 g for about 1 min. The amount of the paclitaxel in thesupernatant was then analyzed by HPLC. HPLC analysis was performed usinga 110A pump and C-8 ultrasphere column (Beckman), and a SPD-6A uvdetector set at 232 nm, a SIL-9A autoinjector and a C—R3A integrator(Shimadzu). The injection volume was 20 μl and the flow rate was 1ml/min. The mobile phase was 58% acetonitrile, 5% methanol, and 37%distilled water.

E. In Vivo Animal Studies

Ten week old DBA/2j female mice were acclimatized for 3-4 days afterarrival. Each mouse was injected subcutaneously in the posterior lateralflank with 10×10⁵ MDAY-D2 tumor cells in 100 μl of PBS on day 1. On day6, the mice were randomly divided into two groups. Group 1 wereimplanted with paste alone (control), and group 2 were implanted withpaste loaded with paclitaxel. A subcutaneous pocket near the tumor wassurgically formed under anaesthesia and approximately 100 mg of moltenpaste (warmed to 50° C.-60° C.) was placed in the pocket and the woundclosed. On day 16, the mice were sacrificed, and the tumors were removedand weighed. Day 16 was selected to allow the tumor growing into aeasily measurable size within the ethical limit.

F. Results and Discussion

The molecular weight and molecular weight distribution ofPDLLA-PEG-PDLLA, relative to polystyrene standards, were measured by GPC(FIG. 21). The intrinsic viscosity of the copolymer in CHCl₃ at 25° C.was determined using a Canon-Fenske viscometer. The molecular weight andintrinsic viscosity decreased with increasing PEG content. Thepolydispersities of PDLLA-PEG-PDLLA with PEG contents of 10%-40% werefrom 2.4 to 3.5. However, the copolymer with 70% PEG had a narrowmolecular weight distribution with a polydispersity of 1.21. This mightbe because a high PEG content reduced the chance of side reactions suchas transesterfication which results in a wide distribution of polymermolecular weight. Alternatively, a coiled structure of thehydrophobic-hydrophilic block copolymers may result in an artificial lowpolydispersity value.

DSC scans of pure PEG and PDLLA-PEG-PDLLA copolymers are given in FIGS.21 and 22. The PEG and PDLLA-PEG-PDLLA with PEG contents of 70% and 40%showed endothermic peaks with decreasing enthalpy and temperature as thePEG content of the copolymer decreased. The endothermic peaks in thecopolymers of 40% and 70% PEG were probably due to the melting of thePEG region, indicating the occurrence of phase separation. While purePEG had a sharp melting peak, the copolymers of both 70% and 40% PEGshowed broad peaks with a distinct shoulder in the case of 70% PEG. Thebroad melting peaks may have resulted from the interference of PDLLAwith the crystallization of PEG. The shoulder in the case of 70% PEGmight represent the glass transition of the PDLLA region. No thermalchanges occurred in the copolymers with PEG contents of 10%, 20% and 30%in a temperature range of 10-250° C., indicating that no significantcrystallization (therefore may be the phase separation) had occurred.

DSC thermograms of PDLLA:PCL (70:30, 80:20, 90:10) blends withoutpaclitaxel or with 20% paclitaxel showed an endothermic peak at about60° C., resulting from the melting of PCL. Due to the amorphous natureof the PDLLA and its low molecular weight (800), melting and glasstransitions of PDLLA were not observed. No thermal changes due to therecrystallization or melting of paclitaxel was observed.

PDLLA-PEG-PDLLA copolymers of 20% and 30% PEG content were selected asoptimum formulation materials for the paste for the following reasons.PDLLA-PEG-PDLLA of 10% PEG could not be melted at a temperature of about60° C. The copolymers of 40% and 70% PEG were readily melted at 60° C.,and the 20% and 30% PEG copolymer became a viscous liquid between 50° C.to 60° C. The swelling of 40% and 70% PEG copolymers in water was veryhigh resulting in rapid dispersion of the pastes in water.

The in vitro release profiles of paclitaxel from PDLLA-PEG-PDLLAcylinders are shown in FIG. 23. The experiment measuring release fromthe 40% PEG cylinders was terminated since the cylinders had a very highdegree of swelling (about 200% water uptake within one day) anddisintegrated in a few days. The released fraction of paclitaxel fromthe 30% PEG cylinders gradually increased over 70 days. The releasedfraction from the 20% PEG cylinders slowly increased up to 30 days andthen abruptly increased, followed by another period of gradual increase.A significant difference existed in the extent to which each individualcylinder (20% PEG content) showed the abrupt change in paclitaxelrelease. Before the abrupt increase, the release fraction of paclitaxelwas lower for copolymers of lower PEG content at the same cylinderdiameter (1 mm). The 40% and 30% PEG cylinders showed much higherpaclitaxel release rates than the 20% PEG cylinders. For example, thecylinder of 30% PEG released 17% paclitaxel in 30 days compared to a 2%release from the 20% PEG cylinder. The cylinders with smaller diametersresulted in faster release rates, e.g., in 30 days, the 30% PEGcylinders with 0.65 mm and 1 mm diameters released 26% and 17%paclitaxel, respectively (FIG. 23).

The above observations may be explained by the release mechanisms ofpaclitaxel from the cylinders. Paclitaxel was dispersed in the polymeras crystals as observed by optical microscopy. The crystals begandissolving in the copolymer matrix at 170° C. and completely dissolvedat 180° C. as observed by hot stage microscope. DSC thermograms of 20%paclitaxel loaded PDLLA-PEG-PDLLA (30% PEG) paste revealed a smallrecrystallization exotherm (16 J/g, 190° C.) and a melting endotherm (6J/g, 212° C.) for paclitaxel (FIG. 21) indicating the recrystallizationof paclitaxel from the copolymer melt after 180° C. In this type ofdrug/polymer matrix, paclitaxel could be released via diffusion and/orpolymer erosion.

In the diffusional controlled case, drug may be released by moleculardiffusion in the polymer and/or through open channels formed byconnected drug particles. Therefore at 20% loading, some particles ofpaclitaxel were isolated and paclitaxel may be released by dissolutionin the copolymer followed by diffusion. Other particles of paclitaxelcould form clusters connecting to the surface and be released throughchannel diffusion. In both cases, the cylinders with smaller dimensiongave a faster drug release due to the shorter diffusion path (FIG. 23).

The dimension changes and water uptake of the cylinders were recordedduring the release (FIG. 24). The changes in length, diameter and wetweight of the 30% PEG cylinders increased rapidly to a maximum within 2days, remained unchanged for about 15 days, then decreased gradually.The initial diameter of the cylinder did not affect the swellingbehavior. For the cylinder of 20% PEG, the length decreased by 10% inone day and leveled off, while the diameter and water uptake graduallyincreased over time. Since more PEG in the copolymer uptaken more waterto facilitate the diffusion of paclitaxel, a faster release was observed(FIG. 23).

The copolymer molecular weight degradation of PDLLA-PEG-PDLLA paste wasmonitored by GPC. For the 20% PEG cylinder, the elution volume at thepeak position increased with time indicating a reduced polymer molecularweight during the course of the release experiment (FIG. 25). A biphasicmolecular weight distribution was observed at day 69. Polymer molecularweight was also decreased for 30% PEG cylinders (1 mm and 0.65 mm).However no biphasic distribution was observed.

NMR spectra revealed a PEG peak at 3.6 ppm and PDLLA peaks at 1.65 ppmand 5.1 ppm. The peak area of PEG relative to PDLLA in the copolymerdecreased significantly after 69 days (FIG. 26), indicating thedissolution of PEG after its dissociation from PDLLA. The dry mass lossof the cylinders was also recorded (FIG. 26) and shows a degradationrate decreasing in the order 30% PEG-0.65 mm>30% PEG-1 mm>20% PEG-1 mm.

The morphological changes of the dried cylinders before and duringpaclitaxel release were observed using SEM (FIG. 27). Briefly, solidpaclitaxel crystals and non-porous polymer matrices were seen before therelease (FIGS. 27A and 27B). After 69 days of release, no paclitaxelcrystals were observed and the matrices contained many pores due topolymer degradation and water uptake (FIGS. 27C and 27D).

The 30% PEG cylinders showed extensive swelling after only two days inwater (FIG. 24) and therefore the hindrance to diffusion of the detachedwater soluble PEG block and degraded PDLLA (i.e., DL-lactic acidoligomers) was reduced. Since the mass loss and degradation of the 30%PEG cylinders was continuous, the contribution of erosion releasegradually increased resulting in a sustained release of paclitaxelwithout any abrupt change (FIG. 23). vFor the 20% PEG cylinders, theswelling was low initially (FIG. 24) resulting in a slow diffusion ofthe degradation products. Therefore the degradation products in theinterior region are primarily retained while there are much lessdegradation products in the outer region due to the short diffusionpath. The degradation products accelerated the degradation rate sincethe carboxylic acid end groups of the oligomers catalyzed the hydrolyticdegradation. This results in a high molecular weight shell and a lowmolecular weight interior as indicated by the biphasic copolymermolecular weight distribution (FIG. 25, day 69). Since the shell rupturewas dependent on factors such as the strength, thickness and defects ofthe shell and interior degradation products, the onset and the extent ofthe loss of interior degradation products are very variable. Because theshell rupture is not consistent and the drug in the polymer is notmicroscopically homogenous, the time point for the release burst and theextent of the burst were different for the 4 samples tested (FIG. 23).

The release of paclitaxel from PDLLA and PCL blends and pure PCL areshown in FIG. 28. Briefly, the released fraction increased with PDLLAcontent in the blend. For example, within 10 days, the releasedpaclitaxel from 80:20, 70:30, and 0:100 PDLLA:PCL were 17%, 11%, and 6%,respectively. After an initial burst in one day, approximately constantrelease was obtained from 80:20 PDLLA:PCL paste. No significant degreeof swelling was observed during the release. For the PDLLA:PCL blends,since PDLLA had a very low molecular weight of about 800, it washydrolyzed rapidly into water soluble products without a long delay inmass loss. PCL served as the “holding” material to keep the paste fromrapidly disintegrating. Therefore the release rate increased with PDLLAcontent in the blend due to the enhanced degradation. The continuouserosion of the PDLLA controlled the release of paclitaxel and resultedin a constant release. The release of paclitaxel from pure PCL wasprobably diffusion controlled due to the slow degradation rate (in 1-2years) of PCL.

Difficulties were encountered in the release study for 20% paclitaxelloaded 90:10 PDLLA:PCL paste due to the disintegration of the pastepellet within 24 hours of incubation. Briefly, during the first 12 hoursof incubation, samples were taken every hour in order to ensure sinkconditions for paclitaxel release. The released paclitaxel from the90:10 paste was 25-35% within 10 hours.

The efficacy of the paste formulations for regressing tumor growth inmice were evaluated (FIG. 29). Briefly, pastes examined were PCL±20%paclitaxel, 80:20 PDLLA:PCL±20% paclitaxel, 90:10 PDLLA:PCL±20%paclitaxel and PDLLA-PEG-PDLLA (30% PEG)±20% paclitaxel. The pasteformulations, 90:10 PDLLA:PCL and PDLLA-PEG-PDLLA, containing paclitaxelreduced tumor growth in vivo by 54 and 40%, respectively. In contrast,the paste formulations, PCL and 80:20 PDLLA:PCL, containing paclitaxelhad little or no effect on tumor growth. All control pastes (drugabsent) had no significant effect on tumor growth. The pasteformulations with faster release rates of paclitaxel (90:10 PDLLA:PCLand PDLLA-PEG-PDLLA) were also more effective in reducing tumor growth,suggesting that a critical local concentration of paclitaxel is requiredat the tumor site for tumor growth inhibition. Paste formulationsreleasing paclitaxel slowly, such as PCL and 80:20 PDLLA:PCL, were noteffective. All of the paste formulations examined had no significanteffect on the body weights of mice, indicating that the paclitaxelloaded paste was well tolerated in vivo.

These data suggest that local application of paclitaxel at the tumorsite is an effective therapeutic strategy to inhibit local tumor growthwithout increasing systemic toxicity. The inability to the paclitaxelloaded formulations to completely inhibit tumor growth is most likelydue to insufficient release of paclitaxel from the polymer and rapidtumor growth of MDAY-D2 tumors. The ability of 90:10 PDLLA:PCL pastecontaining 30% paclitaxel, which released more paclitaxel than 90:10PDLLA:PCL paste containing 20% paclitaxel, and which inhibited tumorgrowth more effectively is consistent in this regard. Thus, modulationof the release rate of paclitaxel, which is regulated by the propertiesof the polymer and chemotherapeutic agents as well as the site ofadministration, is in important step in the development of local therapyfor inhibiting tumor growth.

Example 12 Manufacture of Polymeric Compositions Containing PCL andMePEG

A. Paclitaxel Release from PCL

Polycaprolactone containing various concentrations of paclitaxel wasprepared as described in Example 1. The release of paclitaxel over timewas measured by HPLC essentially as described above. Results are shownin FIG. 30.

B. Effect of MePEG on Paclitaxel Release

MePEG at various concentrations was formulated into PCL paste containing20% paclitaxel, utilizing the methods described in Example 1. Therelease of paclitaxel over time was measured by HPLC essentially asdescribed above. Results of this study are shown in FIG. 31.

C. Effect of MePEG on the Melting Point of PCL

MePEG at various concentrations (formulated into PCL paste containing20% paclitaxel) was analyzed for melting point using DSC analysis at aheating rate of 2.5° C. per minute. Results are shown in FIGS. 32A(melting point vs. % MePEG) and 32B (percent increase in time tosolidify vs. % MePEG).

D. Tensile Strength of MePEG Containing PCL

PCL containing MePEG at various concentrations was tested for tensilestrength and time to fail by a CT-40 Mechanical Strength Tester. Resultsare shown in FIG. 33.

E. Effect of γ-irradiation or the Release of Paclitaxel

PCL:MePEG (80:20) paste loaded with 20% paclitaxel was γ-irradiated andanalyzed for paclitaxel release over time. Results are set forth in FIG.34.

In summary, based on the above experiments it can be concluded that theaddition of MePEG makes the polymer less brittle and more wax like,reduces the melting point and increases the solidification time of thepolymer. All these factors improve the application properties of thepaste. At low concentrations (20%) MePEG has no effect on the release ofpaclitaxel from PCL. Gamma-irradiation appears to have little effect onpaclitaxel release.

Example 13 Preparation of PCL Microspheres: Scale Up Studies

Microspheres (50 g) were prepared using PCL (nominal molecular weight80,000) using the solvent evaporation method described below.

A. Method

A preparation of 500 ml of 10% PCL in methylene chloride and a 4000 mlsolution of 1% PVA (mol. Wt 13,000-23,000; 99% hydrolyzed) wereemulsified using the Homo Mixer controlled with a rheostat at 40 settingfor 10 hours. The mixture was strained using sieve #140 until themicrospheres settled at the bottom then supernatant was decanted. Thepreparation was then washed 3× with distilled water (using thesedimentation followed by decanting method) and then re-suspended in 250ml of distilled water and filtered. The microspheres were then air-driedovernight at 37° C.

B. Results

Microsphere yields were as follows: Initial wt of PCL = 50.1 g Wt. Ofmicrospheres obtained = 41.2 g % yield = (43.2/50.0) × 100 = 86.4

Yield (10-50 μm) about 72%

Mean size 21.4 μm, median 22.0 μm mode 24.7 μm.

Narrower size ranges (20-40 μm) can be obtained by sieving or byseparation using the sedimentation method.

Example 14 Manufacture of PLGA Microspheres

Microspheres were manufactured from (PLLA) lactic acid-glycolic acid(GA) copolymers.

A. Method:

Microspheres were manufactured in the size ranges 0.5 to 10 μm, 10-20 μmand 30-100 μm using standard methods (polymer was dissolved indichloromethane and emulsified in a polyvinyl alcohol solution withstirring as previously described in PCL or PDLLA microspheresmanufacture methods). Various ratio's of PLLA to GA were used as thepolymers with different molecular weights [given as Intrinsic Viscosity(I.V.)]

B. Result:

Microspheres were manufactured successfully from the following startingpolymers: PLLA:GA I.V. 50:50 0.74 50:50 0.78 50:50 1.06 65:35 0.55 75:250.55 85:15 0.56

Paclitaxel at 10% or 20% loadings was successfully incorporated into allthese microspheres. Examples of size distributions for one startingpolymer (85:15, IV=0.56) are given in FIGS. 35-38. Paclitaxel releaseexperiments were performed using microspheres of various sizes andvarious compositions. Release rates are shown in FIGS. 39-42.

Example 15 Di-Block Copolymers

Diblock copolymers of poly(DL-lactide)-block-methoxy polyethylene glycol(PDLLA-MePEG), polycaprolactone-block-methoxy polyethylene glycol(PCL-MePEG) and poly(DL-lactide-co-caprolactone)-block-methoxypolyethylene glycol (PDLLACL-MePEG) were synthesized using a bulk meltpolymerization procedure. Briefly, given amounts of monomers DL-lactide,caprolactone, and methoxy polyethylene glycols with different molecularweights were heated (130° C.) to melt under the bubbling of nitrogen andstirring. Catalyst stannous octoate (0.2% w/w) was added to the moltenmonomers. The polymerization was carried out for 4 hours. The molecularweights, critical micelle concentrations, and the maximum paclitaxelloadings were measured with GPC, fluorescence, and solubilizationtesting, respectively (FIG. 43). High paclitaxel carrying capacitieswere obtained. The ability of solubilizing paclitaxel depends on thecompositions and concentrations of the copolymers (FIGS. 43 and 44).PDLLA-MePEG gave the most stable solubilized paclitaxel (FIGS. 44 and45).

Example 16 Encapsulation of Paclitaxel in Nylon Microcapsules

A. Preparation of Paclitaxel-Loaded Microcapsules

Paclitaxel was encapsulated into nylon microcapsules using theinterfacial polymerization techniques. Briefly, 100 mg of Paclitaxel and100 mg of Pluronic F-127 was dissolved in 1 ml of dichloromethane (DCM)and 0.4 ml (about 500 mg) of adipoyl chloride (ADC) was added. Thissolution was homogenized into 2% PVA solution using the Polytronhomogenizer (1 setting) for 15 seconds. A solution of 1,6-hexane-diamine(HMD) in 5 ml of distilled water was added dropwise while homogenizing.The mixture was homogenized for a further 10 seconds after the additionof HMD solution. The mixture was transferred to a beaker and stirredwith a magnetic stirrer for 3 hours. The mixture was centrifuged,collected and resuspended in 1 ml distilled water.

B. Encapsulation Efficiency/Paclitaxel-Loading

About 0.5 ml of the suspension was filtered and the microspheres weredried. About 2.5 mg of the microcapsules was weighed and suspended in 10ml of acetonitrile for 24 hours. The supernatant analyzed for paclitaxeland the result was expressed as a percentage of paclitaxel. Preliminarystudies have shown that paclitaxel could be encapsulated in nylonmicrocapsules at a high loading (up to 60%) and high encapsulationefficiency (greater than 80%).

C. Paclitaxel Release Studies

About 2.5 mg of the paclitaxel-nylon microspheres were suspended in 50ml water containing 1M each of sodium chloride and urea and analyzedperiodically. Release of paclitaxel from the microcapsule was fast withmore than 95% of the drug released after 72 hours. (FIG. 46).

Example 17 Complexation of Paclitaxel with Cyclodextrins

A. Materials

Paclitaxel was obtained from Hauser Chemicals Inc., Boulder, Colo.Disodium phosphate (Fisher), citric acid (British Drug Houses),Hydroxypropyl-β-cyclodextrin (HPβCD), γ-cyclodextrin (γ-CD) andhydroxypropyl-γ-cyclodextrin (HPγCD) were obtained from AmericanMaize-Products Company (Hammond, Ind.) and were used as received.

B. Methods

1. Solubility Studies

Excess amounts of paclitaxel (5 mg) were added to aqueous solutionscontaining various concentrations of γ-CD, HPγ-CD, or HPβ-CD and tumbledgently for about 24 hours at 37° C. After equilibration, aliquots of thesuspension were filtered through a 0.45 μm membrane filter (Millipore),suitably diluted and analyzed using HPLC. The mobile phase was composedof a mixture of acetonitrile, methanol and water (58:5:37) at a flowrate of 1.0 ml min⁻¹. The solubility of paclitaxel in a solvent composedof 50:50 water and ethanol (95%) containing various concentrations, upto 10%, of HPβ-CD was also investigated. In addition, dissolution rateprofiles of paclitaxel were investigated by adding 2 mg of paclitaxel(as received) to 0, 5, 10 or 20% HPγ-CD solutions or 2 mg of previouslyhydrated paclitaxel (by suspending in water for 7 days) to pure waterand tumbling gently at 37° C. Aliquots were taken at various timeintervals and assayed for paclitaxel.

2. Stability Studies

The solutions containing 20% HPβCD or HPγCD had pH values of 3.9 and5.2, respectively. The stability of paclitaxel in cyclodextrin solutionswas investigated by assaying paclitaxel in solutions (20 μg ml⁻¹)containing 10 or 20% HPγ-CD or HPβ-CD in either water or a 50:50water-ethanol mixture at 37° C. or 55° C. at various time intervals. Inaddition, stability of paclitaxel in solutions (1 μg/ml) containing 1%,2% or 5% HPβCD at 55° C. were determined.

C. Results

1. Solubility Studies

The solubility of paclitaxel increased over the entire CD concentrationrange studied; HPβCD producing the greatest increase in the solubilityof paclitaxel (FIG. 47). The shape of the solubility curves suggeststhat the stoichiometries were of higher order than a 1:1 complex.Paclitaxel formed Type A_(P) curves with both HPβCD and HPγCD and TypeA_(N) curves with γCD. The solubility of paclitaxel in a 50% solution ofHPβCD in water was 3.2 mg ml⁻ at 37° C. which was about a 2000-foldincrease over the solubility of paclitaxel in water. The estimatedstability constants (from FIG. 48) for first order complexes ofpaclitaxel-cyclodextrins were 3.1, 5.8 and 7.2 M⁻¹ for γ-CD, HPγCD andHPβCD and those for second order complexes were 0.785×10³, 1.886×10³ and7.965×10³ M⁻¹ for ₇—CD, HPγCD and HPβCD respectively. The values of theobserved stability constants suggested that the inclusion complexesformed by paclitaxel with cyclodextrins were predominantly second ordercomplexes.

The solubility of paclitaxel in 50:50 water:ethanol mixture increasedwith an increase in the cyclodextrin concentration (FIG. 49) as observedfor complexation in pure water. The apparent stability constant for thecomplexation of paclitaxel and HPβCD in the presence of 50% ethanol(26.57 M⁻¹) was significantly lower (about 300 times) than the stabilityconstant in the absence of ethanol. The lower stability constant may beattributed to a change in the dielectric constant or the polarity of thesolvent in the presence of ethanol.

The dissolution profiles of paclitaxel in 0, 5, 10 and 20% γCD solutions(FIG. 50) illustrates the formation of a metastable solution ofpaclitaxel in pure water or the cyclodextrin solutions; the amount ofpaclitaxel in solution gradually increased, reached a maximum andsubsequently decreased. Dissolution studies using paclitaxel sampleswhich were previously hydrated by suspending in water for 48 hours didnot show the formation of the metastable solution. In addition, DSCanalysis of the hydrated paclitaxel (dried in a vacuum oven at roomtemperature) showed two broad endothermic peaks between 60 and 110° C.These peaks were accompanied by about 4.5% weight loss (determined bythermogravimetric analysis) indicating the presence of hydrate(s). Aloss in weight of about 2.1% would suggest the formation of a paclitaxelmonohydrate. Therefore, the occurrence of the DSC peaks between 60° C.and 110° C. and the loss in weight of about 4.5% suggests the presenceof a dihydrate. There was no evidence of endothermic peak(s) between 60°C. and 110° C. (DSC results) or a weight loss (TGA results) forpaclitaxel samples as received. Therefore, (as received) paclitaxel wasanhydrous and on suspension in water it dissolved to form asupersaturated solution which recrystallized as a hydrate of lowersolubility (FIG. 50).

2. Stability Studies

Paclitaxel degradation depended on the concentration of the cyclodextrinand followed pseudo-first order degradation kinetics (e.g., FIG. 51).The rate of degradation of paclitaxel in solutions (1 μg/ml paclitaxel)containing 1% HPβCD at 55° C. faster (k=3.38×10⁻³ h⁻¹) than the rate athigher cyclodextrin concentrations. Degradation rate constants of1.78×10⁻³ h⁻¹ and 0.96×10⁻³ h⁻¹ were observed for paclitaxel in 10%HPβCD and HPγCD, respectively. Paclitaxel solutions (1 μg/ml) containing2, 4, 6 or 8% HPβCD did not show any significant difference in the rateof degradation from that obtained with the 10 or 20% HPβCD solutions (20μg/ml). The presence of ethanol did not adversely affect the stabilityof paclitaxel in the cyclodextrin solutions.

D. Conclusion

This study showed that the solubility of paclitaxel could be increasedby complexation with cyclodextrins. These aqueous based cyclodextrinformulations may have potential for peritubular in the treatment ofvarious cancers.

Example 18 Polymeric Compositions with Increased Concentrations ofPaclitaxel

PDLLA-MePEG and PDLLA-PEG-PDLLA are block copolymers with hydrophobic(PDLLA) and hydrophilic (PEG or MePEG) regions. At appropriate molecularweights and chemical composition, they may form tiny aggregates ofhydrophobic PDLLA core and hydrophilic MePEG shell. Paclitaxel can beloaded into the hydrophobic core, thereby providing paclitaxel with anincreased “solubility”.

A. Materials

D,L-lactide was purchased from Aldrich, Stannous octoate, poly (ethyleneglycol) (mol. wt. 8,000), MePEG (mol. wt. 2,000 and 5,000) were fromSigma. MePEG (mol. wt. 750) was from Union Carbide. The copolymers weresynthesized by a ring opening polymerization procedure using stannousoctoate as a catalyst (Deng et al, J. Polym. Sci., Polym, Lett.28:411-416, 1990; Cohn et al, J. Biomed, Mater. Res. 22: 993-1009,1988).

For synthesizing PDLLA-MePEG, a mixture of DL-lactide/MePEG/stannousoctoate was added to a 10 milliliter glass ampoule. The ampoule wasconnected to a vacuum and sealed with flame. Polymerization wasaccomplished by incubating the ampoule in a 150° C. oil bath for 3hours. For synthesizing PDLLA-PEG-PDLLA, a mixture ofD,L-lactide/PEG/stannous octoate was transferred into a glass flask,sealed with a rubber stopper, and heated for 3 hours in a 150° C. oven.The starting compositions of the copolymers are given in Tables II andIII. In all the cases, the amount of stannous octoate was 0.5%-0.7%.

B. Methods

The polymers were dissolved in acetonitrile and centrifuged at 10,000 gfor 5 minutes to discard any non-dissolvable impurities. Paclitaxelacetonitrile solution was then added to each polymer solution to give asolution with paclitaxel (paclitaxel+polymer) of 10%-wt. The solventacetonitrile was then removed to obtain a clear paclitaxel/PDLLA-MePEGmatrix, under a stream of nitrogen and 60° C. warming. Distilled water,0.9% NaCl saline, or 5% dextrose was added at four times weight of thematrix. The matrix was finally “dissolved” with the help of vortexmixing and periodic warming at 60° C. Clear solutions were obtained inall the cases. The particle sizes were all below 50 nm as determined bya submicron particle sizer, NICOMP Model 270. The formulations are givenin Table II. TABLE II Formulations of Paclitaxel/PDLLA-MePEG* PaclitaxelLoading (final PDLLA-MePEG Dissolving Media paclitaxel concentrate)2000/50/50 water 10% (20 mg/ml) 2000/40/60 water 10% (20 mg/ml)2000/50/50 0.9% saline  5% (10 mg/ml) 2000/50/50 0.9% saline 10% (20mg/ml) 2000/50/50   5% dextrose 10% (10 mg/ml) 2000/50/50   5% dextrose10% (20 mg/ml)

In the case of PDLLA-PEG-PDLLA (Table III), since the copolymers cannotdissolve in water, paclitaxel and the polymer were co-dissolved inacetone. Water or a mixture of water/acetone was gradually added to thispaclitaxel polymer solution to induce the formation ofpaclitaxel/polymer spheres. TABLE III Composition of PDLLA-PEG-PDLLACopolymer Name Wt. of PEG (g) Wt. of DL-lactide (g) PDLLA-PEG-PDLLA 1 990/10 PDLLA-PEG-PDLLA 2 8 80/20 PDLLA-PEG-PDLLA 3 7 70/30PDLLA-PEG-PDLLA 4 6 60/40 PDLLA-PEG-PDLLA 14 6 30-/70*PEG molecular weight. 8,000.

C. Results

Many of the PDLLA-MePEG compositions form clear solutions in water, 0.9%saline, or 5% dextrose, indicating the formation of tiny aggregates inthe range of nanometers. Paclitaxel was loaded into PDLLA-MePEGnanoparticles successfully. For example, at % loading (this represents10 mg paclitaxel in 1 ml paclitaxel/PDLLA-MePEG/aqueous system), a clearsolution was obtained from 2000-50/50 and 2000-40/60. The particle sizewas about 20 nm.

Example 19 Analysis of Drug Release

A known weight of a polymer (typically a 2.5 mg pellet) is added to a 15ml test tube containing 14 ml of a buffer containing 10 mmNa₂HPO₄—NaH₂PO₄, 0.145 m NaCl and 0.4 g/l bovine serum albumin. Thetubes are capped and tumbled at 37° C. At specific times all the 14 mlof the liquid buffer are removed and replaced with fresh liquid buffer.

The liquid buffer is added to 1 milliliter of methylene chloride andshaken for 1 minute to extract all the paclitaxel into the methylenechloride. The aqueous phase is then removed and the methylene chloridephase is dried under nitrogen. The residue is then dissolved in 60%acetonitrile: 40% water and the solution is injected on to a HPLC systemusing the following conditions: C8 column (Beckman Instruments USA),mobile phase of 58%:5%:37% acetonitrile: methanol: water at a flow rateof 1 minute per minute.

For paclitaxel the collected buffer is then analyzed at 232 nm. For MTXthe collected buffer is applied directly to the HPLC column with no needfor extraction in methylene chloride. MTX is analyzed at 302 nm. ForVanadium containing compounds the liquid buffer is analyzed directlyusing a UV/VIS spectrometer in the 200 to 300 nm range.

Example 20 Effect of Paclitaxel-Loaded Thermopaste on Tumor Growth andTumor Angiogenesis In Vivo

Fertilized domestic chick embryos are incubated for 3 days prior tohaving their shells removed. The egg contents are emptied by removingthe shell located around the airspace, severing the interior shellmembrane, perforating the opposite end of the shell and allowing the eggcontents to gently slide out from the blunted end. The contents areemptied into round-bottom sterilized glass bowls, covered with petridish covers and incubated at 90% relative humidity and 3% carbondioxide.

MDAY-D2 cells (a murine lymphoid tumor) is injected into mice andallowed to grow into tumors weighing 0.5-1.0 g. The mice are sacrificed,the tumor sites wiped with alcohol, excised, placed in sterile tissueculture media, and diced into 1 mm pieces under a laminar flow hood.Prior to placing the dissected tumors onto the 9-day old chick embryos,CAM surfaces are gently scraped with a 30 gauge needle to insure tumorimplantation. The tumors are then placed on the CAMs after 8 days ofincubation (4 days after deshelling), and allowed to grow on the CAM forfour days to establish a vascular supply. Four embryos are preparedutilizing this method, each embryo receiving 3 tumors. For theseembryos, one tumor receives 20% paclitaxel-loaded thermopaste, thesecond tumor unloaded thermopaste, and the third tumor no treatment. Thetreatments are continued for two days before the results were recorded.

The explanted MDAY-D2 tumors secrete angiogenic factors which induce theingrowth of capillaries (derived from the CAM) into the tumor mass andallow it to continue to grow in size. Since all the vessels of the tumorare derived from the CAM, while all the tumor cells are derived from theexplant, it is possible to assess the effect of therapeuticinterventions on these two processes independently. This assay has beenused to determine the effectiveness of paclitaxel-loaded thermopaste on:(a) inhibiting the vascularization of the tumor and (b) inhibiting thegrowth of the tumor cells themselves.

Direct in vivo stereomicroscopic evaluation and histological examinationof fixed tissues from this study demonstrated the following. In thetumors treated with 20% paclitaxel-loaded thermopaste, there was areduction in the number of the blood vessels which supplied the tumor(see FIGS. 51C and 51D), a reduction in the number of blood vesselswithin the tumor, and a reduction in the number of blood vessels in theperiphery of the tumor (the area which is typically the most highlyvascularized in a solid tumor) when compared to control tumors (FIGS.51A and 51B). The tumors began to decrease in size and mass during thetwo days the study was conducted. Additionally, numerous endothelialcells were seen to be arrested in cell division indicating thatendothelial cell proliferation had been affected. Tumor cells were alsofrequently seen arrested in mitosis. All 4 embryos showed a consistentpattern with the 20% paclitaxel-loaded thermopaste suppressing tumorvascularity while the unloaded thermopaste had no effect.

By comparison, in CAMs treated with unloaded thermopaste, the tumorswere well vascularized with an increase in the number and density ofvessels when compared to that of the normal surrounding tissue, anddramatically more vessels than were observed in the tumors treated withpaclitaxel-loaded paste. The newly formed vessels entered the tumor fromall angles appearing like spokes attached to the center of a wheel (seeFIGS. 51A and 51B). The control tumors continued to increase in size andmass during the course of the study. Histologically, numerous dilatedthin-walled capillaries were seen in the periphery of the tumor and fewendothelial were seen to be in cell division. The tumor tissue was wellvascularized and viable throughout.

As an example, in two similarly-sized (initially, at the time ofexplantation) tumors placed on the same CAM the following data wasobtained. For the tumor treated with 20% paclitaxel-loaded thermopastethe tumor measured 330 mm×597 mm; the immediate periphery of the tumorhas 14 blood vessels, while the tumor mass has only 3-4 smallcapillaries. For the tumor treated with unloaded thermopaste the tumorsize was 623 mm×678 mm; the immediate periphery of the tumor has 54blood vessels, while the tumor mass has 12-14 small blood vessels. Inaddition, the surrounding CAM itself contained many more blood vesselsas compared to the area surrounding the paclitaxel-treated tumor.

This study demonstrates that thermopaste releases sufficient quantitiesof anti-proliferative agent (in this case paclitaxel) to inhibit thepathological angiogenesis which accompanies tumor growth anddevelopment. Under these conditions angiogenesis is maximally stimulatedby the tumor cells which produce angiogenic factors capable of inducingthe ingrowth of capillaries from the surrounding tissue into the tumormass. The 20% paclitaxel-loaded thermopaste is capable of blocking thisprocess and limiting the ability of the tumor tissue to maintain anadequate blood supply. This results in a decrease in the tumor mass boththrough a cytotoxic effect of the drug on the tumor cells themselves andby depriving the tissue of the nutrients required for growth andexpansion.

Example 21 Effect of Therapeutic Agent-Loaded Thermopaste on TumorGrowth In Vivo in a Murine Tumor Model

The murine MDAY-D2 tumor model may be used to examine the effect oflocal slow release of an anti-proliferative compound such as paclitaxelon tumor growth, tumor metastasis, and animal survival. Briefly, theMDAY-D2 tumor cell line is grown in a cell suspension consisting of 5%Fetal Calf Serum in alpha mem media. The cells are incubated at 37° C.in a humidified atmosphere supplemented with 5% carbon dioxide, and arediluted by a factor of 15 every 3 days until a sufficient number ofcells are obtained. Following the incubation period the cells areexamined by light microscopy for viability and then are centrifuged at1500 rpm for 5 minutes. PBS is added to the cells to achieve a dilutionof 1,000,000 cells per ml.

Ten week old DBA/2j female mice are acclimatized for 3-4 days afterarrival. Each mouse is then injected subcutaneously in theposteriolateral flank with 100,000 MDAY-D2 cells in 100 ml of PBS.Previous studies have shown that this procedure produces a visible tumorat the injection site in 3-4 days, reach a size of 1.0-1.7 g by 14 days,and produces visible metastases in the liver 19-25 days post-injection.Depending upon the objective of the study a therapeutic intervention canbe instituted at any point in the progression of the disease.

Using the above animal model, 20 mice are injected with 140,000 MDAY-D2cells s.c. and the tumors allowed to grow. On day 5 the mice are dividedinto groups of 5. The tumor site was surgically opened under anesthesia,the local region treated with the drug-loaded thermopaste or controlthermopaste without disturbing the existing tumor tissue, and the woundwas closed. The groups of 5 received either no treatment (wound merelyclosed), polymer (PCL) alone, 10% paclitaxel-loaded thermopaste, or 20%paclitaxel-loaded thermopaste (only 4 animals injected) implantedadjacent to the tumor site. On day 16, the mice were sacrificed, thetumors were dissected and examined (grossly and histologically) fortumor growth, tumor metastasis, local and systemic toxicity resultingfrom the treatment, effect on wound healing, effect on tumorvascularity, and condition of the paste remaining at the incision site.

The weights of the tumors for each animal is shown in Table IV below:TABLE IV Tumor Weights (gm) Animal No. Control Control 10% Paclitaxel(empty) (PCL) Thermopaste Thermopaste 20% Paclitaxel 1 1.387 1.137 0.4870.114 2 0.589 0.763 0.589 0.192 3 0.461 0.525 0.447 0.071 4 0.606 0.2820.274 0.042 5 0.353 0.277 0.362 Mean 0.6808 0.6040 0.4318 0.1048 Std.Deviation 0.4078 0.3761 0.1202 0.0653 P Value 0.7647 0.358 0.036Thermopaste loaded with 20% paclitaxel reduced tumor growth by over 85%(average weight 0.105) as compared to control animals (average weight0.681). Animals treated with thermopaste alone or thermopaste containing10% paclitaxel had only modest effects on tumor growth; tumor weightswere reduced by only 10% and 35% respectively (FIG. 52A). Therefore,thermopaste containing 20% paclitaxel was more effective in reducingtumor growth than thermopaste containing 10% paclitaxel (see FIG. 52C;see also FIG. 52B).

Thermopaste was detected in some of the animals at the site ofadministration. Polymer varying in weight between 0.026 g to 0.078 g wasdetected in 8 of 15 mice. Every animal in the group containing 20%paclitaxel-loaded thermopaste contained some residual polymer suggestingthat it was less susceptible to dissolution. Histologically, the tumorstreated with paclitaxel-loaded thermopaste contained lower cellularityand more tissue necrosis than control tumors. The vasculature wasreduced and endothelial cells were frequently seen to be arrested incell division. The paclitaxel-loaded thermopaste did not appear toaffect the integrity or cellularity of the skin or tissues surroundingthe tumor. Grossly, wound healing was unaffected.

Example 22 Use of Paclitaxel Loaded Surgical Paste to Delay Regrowth ofPartially Resected RIF-1 Tumors in Mice

The effectiveness of a biodegradable polymeric sustained releasesurgical paste formulation of paclitaxel in delaying regrowth ofpartially resected RIF-1 tumors in C3H/HeJ mice was investigated.

A. Methods

Paclitaxel (20%) was incorporated into a 4:1 blend ofpoly(ε-caprolactone) and methoxypolyethylene glycol. The in vitrorelease profile for this formulation in phosphate buffered salinecontaining albumin (0.4 mg/mL) at 37° C. was investigated using an HPLCassay for paclitaxel. Briefly, seventeen mice were injected in the rightflank with 100 μl of a RIF-1 cell suspension (1.0×10⁶ cells) in HanksBuffer. The tumors were allowed to grow for 5 days following which morethan 70% of each tumor was surgically resected and the remaining tumormass was left untreated or coated with 20-30 μL of either 20% paclitaxelin surgical paste or surgical paste alone (no drug). This was day 0.Dimensions of the visible tumor mass beneath the skin were measured ondays 4 through 7 and day 9. The area of this visible cylindrical tumorsurface was shown to be correlated to tumor volume (r=0.812).

B. Results

The in vitro release curve of paclitaxel was characterized by an initial1 day burst phase followed by a long period of slow sustained release.The areas of the visible cylindrical tumor surfaces from each mouse fromdays 4 through 9 are shown in Table V. All the mice except 1 in each ofthe two groups which did not receive paclitaxel showed extensive tumorregrowth by day 4. One mouse which received polymer only showed delayedtumor regrowth while one mouse which received no treatment showed noregrowth. In contrast all but one of the mice treated with paclitaxelsurgical paste showed no tumor regrowth until at least day 5 and twomice still did not show regrowth until day 6.

C. Conclusion

Paclitaxel loaded paste significantly inhibited tumor regrowth betweendays 1 to 5. Regrowth occurred after day 6 due to the aggressiveness ofthe cell line. TABLE V RIF-1 cell tumor size (expressed as area ofvisible tumor mass beneath the skin in mm²) measured on days followingtumor resection surgery Tumor sizes (mm²) Treatment day 4 day 5 day 6day 7 day 9 20% paclitaxel in 0.0 0.0 41.3 58.8 * polymer 20% paclitaxelin 30.7 35.3 49.0 67.9 * polymer 20% paclitaxel in 0.0 0.0  0.0 * 42.4polymer 20% paclitaxel in 0.0 47.2 55.4 47.8 * polymer 20% paclitaxel in0.0 25.5 41.9 57.1 * polymer 20% paclitaxel in 0.0 0.0  0.0 * 46.6polymer polymer only 44.2 52.8 * * * polymer only 36.3 39.0 32.7 * 38.5polymer only 36.9 44.2 51.5 42.7 * polymer only 0.0 12.9 14.9 * 29.2polymer only 37.4 39.6 40.7 54.1 * polymer only 40.7 24.2 40.7 * 36.9 notreatment 0.0 0.0  0.0 * * no treatment 52.2 77.8 * * * no treatment39.0 45.4 43.6 * * no treatment 22.1 21.2 31.2 * 26.9 no treatment 21.230.7 36.3 * ** Tumor not measured or animal already sacrificed

Example 23 Encapsulation of Suramin

One milliliter of 5% ELVAX (poly(ethylene-vinyl acetate) cross-linkedwith 5% vinyl acetate) in dichloromethane (“DCM”) is mixed with a fixedweight of sub-micron ground sodium suramin. This mixture is injectedinto 5 ml of 5% Polyvinyl Alcohol (“PVA”) in water in a 30 ml flatbottomed test tube. Tubes containing different weights of the drug arethen suspended in a multi-sample water bath at 40° C. for 90 minuteswith automated stirring. The mixtures are removed, and microspheresamples taken for size analysis. Tubes are centrifuged at 1000 g for 5min. The PVA supernatant is removed and saved for analysis(nonencapsulated drug). The microspheres are then washed (vortexed) in 5ml of water and recentrifuged. The 5 ml wash is saved for analysis(surface bound drug). Microspheres are then wetted in 50 ul of methanol,and vortexed in 1 ml of DCM to dissolve the ELVAX. The microspheres arethen warmed to 40° C., and 5 ml of 50° C. water is slowly added withstirring. This procedure results in the immediate evaporation of DCM,thereby causing the release of sodium suramin into the 5 ml of water.

All samples were assayed for drug content by quantification offluorescence. Briefly, sodium suramin absorbs uv/vis with a lambda maxof 312 nm. This absorption is linear in the 0 to 100 ug/ml range in bothwater and 5% PVA. Sodium suramin also fluoresces strongly with anexcitation maximum at 312 nm, and emission maximum at 400 nm. Thisfluorescence is quantifiable in the 0 to 25 ug/ml range.

The results of these experiments is shown in FIGS. 53-59. Briefly, thesize distribution of microspheres by number (FIG. 53) or by weight (FIG.54) appears to be unaffected by inclusion of the drug in the DCM. Goodyields of microspheres in the 20 to 60 μm range may be obtained.

The encapsulation of suramin is very low (<1%) (see FIG. 56). However asthe weight of drug is increased in the DCM the total amount of drugencapsulated increased although the % encapsulation decreased. As isshown in FIG. 55, 50 ug of drug may be encapsulated in 50 mg of ELVAX.Encapsulation of sodium suramin in 2.5% PVA containing 10% NaCl is shownin FIG. 57 (size distribution by weight). Encapsulation of sodiumsuramin in 5% PVA containing 10% NaCl is shown in FIGS. 58 and 59 (sizedistribution by weight, and number, respectively).

To assess suramin and cortisone acetate as potential anti-angiogenicagents, each agent was mixed with 0.5% methylcellulose and applied thedried disks containing the agent onto the developing blood vessels ofthe 6-day old CAM. A combination treatment of suramin (70 μg) withcortisone acetate (20 μg) was successful in inhibiting angiogenesis whentested on the CAM for 48 hours. The resulting avascular region measured6 mm in diameter and revealed an absence of blood flow and theappearance of sparse blood islands (FIGS. 60A and 60B).

Example 24 Methotrexate-Loaded Paste

A. Manufacture of Methotrexate-Loaded Paste

Methotrexate (“MTX”; Sigma Chemical Co.) is ground in a pestle andmortar to reduce the particle size to below 5 microns. It is then mixedas a dry powder with polycaprolactone (molecular wt 18000 BirminghamPolymers, AL USA). The mixture is heated to 65° C. for 5 minutes and themolten polymer/methotrexate mixture is stirred into a smooth paste for 5minutes. The molten paste is then taken into a 1 mL syringe, andextruded as desired.

B. Results

Results are shown in FIGS. 61A-E. Briefly, FIG. 61A shows MTX releasefrom PCL discs containing 20% MePEG and various concentrations of MTX.FIG. 61B shows a similar experiment for paste which does not containMePEG. FIGS. 61C, D, and E show the amount of MTX remaining in the disk.

As can be seen by the above results, substantial amounts of MTX can bereleased from the polymer when high MePEG concentrations are utilized.

Example 25 Manufacture of Microspheres Containing Methotrexate

A. Microspheres with MTX Alone

Methotrexate (Sigma) was ground in a pestle and mortar to reduce theparticle size to below 5 microns. One hundred milliliters of a 2.5% PVA(w/v) (Aldrich or Sigma) in water was stirred for 15 minutes with 500 mgof unground MTX at 25° C. to saturate the solution with MTX. Thissolution was then centrifuged at 2000 rpm to remove undissolved MTX andthe supernatant used in the manufacture of microspheres.

Briefly, 10 ml of a 5% w/v solution of poly(DL) lactic acid (molecularweight 500,000; Polysciences), Polylactic:glycolic acid (50:50 IV 0.78polysciences) or polycaprolactone (molecular weight 18,000, BPI)containing 10:90 w/w MTX(ground):POLYMER were slowly dripped into 100 mLof the MTX saturated 2.5% w/v solution of PVA (Aldrich or Sigma) withstirring at 600 rpm. The mixture was stirred at 25° C. for 2 hours andthe resulting microspheres were washed and dried.

Using this method MTX loaded microspheres can be reproduciblymanufactured in the 30 to 160 micron range (see FIG. 62).

B. Microspheres with MTX and Hyaluronic Acid

MTX loaded microspheres can be made using hyaluronic acid (“HA”) as thecarrier by a water in oil emulsion manufacture method, essentially asdescribed below. Briefly, 50 ml of Parafin oil (light oil; FisherScientific) is warmed to 60° C. with stirring at 200 rpm. A 5 mLsolution of sodium hyaluronate (20/mL); source=rooster comb; Sigma) inwater containing various amounts MTX is added dropwise into the Parafinoil. The mixture is stirred at 200 rpm for 5 hours, centrifuged at 500×gfor 5 minutes. The resulting microspheres are washed in hexane fourtimes, and allowed to dry.

Example 26 Manufacture of Polymeric Compositions Containing VanadiumCompounds

A. Polymeric Paste Containing Vanadyl Sulfate

Vanadyl Sulfate (Fisher Scientific) is first ground in a pestle andmortar to reduce the particle size, then dispersed into melted PCL asdescribed above for MTX. It is then taken up into a syringe to solidifyand is ready for use.

Drug release was determined essentially as described above in Example33, except that a 65 mg pellet of a 10% W/W VOSO₄:PCL was suspended in10 ml of water and the supernatant analyzed for released VanadylSulphate using UV/Vis absorbance spectroscopy of the peak in the 200 to300 nm range.

Results are shown in FIG. 63. Briefly, from a polymeric compositioncontaining 10% VOSO₄, 1 mg of VOSO₄ was released in 6 hours, 3 mg after2 days and 5 mg by day 6.

B. Polymeric Microspheres Containing Vanadyl Sulfate

Vanadyl sulfate was incorporated into microspheres of polylactic acid orhyaluronic acid essentially as described in Example 25. Results areshown in FIG. 64.

C. Polymeric Paste Containing Organic Vanadate

Organic vanadate is loaded into a PCL paste essentially as describedabove in Example 24. Vanadate release from the microspheres wasdetermined as described above. Results are shown in FIGS. 65A and 65B.

D. Organic Vanadate Containing Microspheres

Organic vanadate may also be loaded into microspheres essentially asdescribed in Example 25. Such microspheres are shown in FIG. 66 for polyD,L lactic acid (M.W. 500,000; Polysciences).

Example 27 Polymeric Composition Containing Bis(Maltolato) Oxovanadium(BMOV)

A. Manufacture of bis(maltolato) Oxovanadium Loaded Paste

Poly (ε-caprolactone) (Molecular weight 20000) (BPI Birmingham Ala.) andBMOV were weighed directly into a glass beaker in the appropriateproportions. In some formulations methoxypolyethylene glycol (MEPEG)(molecular weight 350) (Union Carbide, Danbury Conn.) was also added tothe PCL and BMOV. The beaker and contents were warmed to 55° C. withgentle stirring for 5 minutes until the BMOV was thoroughly dispersed inthe molten polymer. The molten mix was then drawn into a prewarmedsyringe and stored at 4° C. until use.

B. Drug Release Studies

To tubes containing 15 ml of 10 mM phosphate buffered saline (PBS pH7.4) and 100 ug/ml bovine serum albumin (fraction 5 Boehringer Mannheim,Germany) were added 150 mg disc-shaped slabs of PCL-BMOV paste. Thetubes were sealed and tumbled end over end at 30 rpm at 37° C. Atappropriate times the PCL-BMOV slab was allowed to settle under gravityfor 5 minutes and all the supernatant was removed. The BMOVconcentration was determined in the supernatants by measuring theabsorbance at 256 nm (A256) and 276 nm (A276). The supernatant wasreplaced with 15 ml of fresh PBS and the tubes were retumbled. A linearcalibration curve of BMOV concentration vs. A256 or A276 was obtainedusing BMOV standards in the 0 to 25 ug/ml range. The absorbance valuesat 256 nm or 276 nm of these standards were shown to be unaffected bystorage in sealed tubes at 37° C. for 2 to 3 days (the same conditionsused for drug release studies).

At the end of the drug release experiments samples of the PCL-BMOVmatrix were assayed for residual drug content by dissolution of a knowndried weight of the matrix in 0.5 ml of dichloromethane (DCM) (Fisher).To this solution was added 50 ml of warm water (50° C.) with mixing toevaporate the DCM leaving the BMOV in water for spectrophotometricanalysis (A256).

C. Scanning Electron Microscopy (SEM)

Samples of the PCL-BMOV matrix that had been used in the 2 month drugrelease experiments were examined using SEM. These samples were comparedwith freshly prepared control samples. Polymer samples were coated(60:40 gold:palladium) (Hummer instruments, Technics, USA) and examinedusing a Hitachi (model F-2300) scanning electron microscope with a IBMdata collection system.

D. Results

The release of BMOV from the PCL matrix is shown in FIGS. 67A and 67B.Increasing the loading of BMOV from 5% to 35% in the PCL matrixincreased the rate of drug release over the two month period (FIG. 67A).At 35% BMOV loading the release rate increased dramatically. The releaseprofiles for the 20% to 30% BMOV loadings showed an initial more rapidphase of drug release in the first two days followed by a controlled,almost zero order release over the next 2 months.

The drug release profiles are also expressed in terms of the % of drugremaining in the pellet (FIG. 67B). Briefly, the % of BMOV remaining inthe PCL matrix was almost identical at all time points for all the BMOVloadings up to (and including) 30% so that between 65% and 80% of theoriginal BMOV was still present in the matrix after two months. (Onlythe data for 25% and 30% BMOV loadings are shown in FIG. 67B forclarity). The percentage of drug remaining in the matrix decreased morerapidly at a loading 35% BMOV so that a little over 50% of the originalBMOV was present in the matrix after one month. In order to verify thecumulative drug release data, samples of the PCL-BMOV matrix wereassayed for remaining drug content at the end of each drug releaseexperiment. The % drug remaining at 70 days as determined by thisresidual assay was as follows: 67%+/−10% (5% BMOV), 56%+/−10% (10%BMOV), 80%+/−20% (15% BMOV), 84%+/−20% (20% BMOV), 85%+/−12% (25% BMOV),77%+/−14% (30% BMOV) and 57%+/−15% (35% BMOV).

FIGS. 68A and 68B show the effect of adding 20% MEPEG to the PCL matrixon the drug release profiles for various loading concentrations of BMOV.The addition of MEPEG to the matrix increases the release rate of BMOVdramatically compared to the release of BMOV from the PCL alone (FIG.67). Greater than 50% of the BMOV was released from the polymer matrixwithin 7 days 1 week at all BMOV loading concentrations. Residualanalysis of the PCL-BMOV-MEPEG pellets gave the following values for the% of BMOV remaining at 7 days in the pellets: 24% (+/−9%), 5% BMOV; 25%(+/−8%), 10% BMOV; 22% (+/−4%), 15% BMOV; 27% (+/−7%), 20% BMOV.

FIG. 69A shows the morphology of the BMOV crystals under highmagnification. FIGS. 69B and 69C show the morphology of the polymer-drugmatrices both before and after the two month drug release study inaqueous buffer. The PCL-BMOV matrices for 15%, 20% and 30% BMOV loadingswere typically smooth on their external faces before the release studyand a representative SEM is shown in FIG. 69B. Following incubation inthe aqueous buffer for two months the external surfaces were rough andpitted.

E. Discussion

This compound was found to release very slowly from the PCL matrix withalmost ideal release characteristics for the maintenance of sustainedconcentrations of vanadium. These characteristics included only a smallburst effect of drug release in the first few days followed by almostzero order release kinetics at most drug loadings (FIG. 67). BMOV isless water soluble than vanadyl sulphate or sodium orthovanadate and thehydrophobicity of the molecule probably increases the affinity of theBMOV molecules for the hydrophobic PCL matrix and decreases the rate ofdrug release into an aqueous incubation medium.

The addition of 20% MEPEG to PCL has been shown to improve the thermalflow properties of the paste by reducing the viscosity of the matrix andthe temperature at which the polymer solidifies. These properties areimportant in applying the paste to a peritubular tumor site as bettercoverage of the site is obtained under these conditions. The addition ofMEPEG to the BMOV-PCL paste matrix has been shown to enhance the releaserates of BMOV in vitro (FIG. 68) at all BMOV loading concentrations (5%to 20%) relative to the release rates from BMOV-PCL (no MEPEG) (FIG.67). The 5% BMOV loaded PCL-MEPEG paste was shown to release between 500an 1000 ug of BMOV per day (which was similar to the release rate from35% BMOV loaded PCL).

Example 28 In Vitro and In Vivo Efficacy of Bis(Maltolato)OxoVanadiumLoaded Thermopaste

BMOV loaded polymeric PCL thermopaste was prepared as describedpreviously and was tested for its efficacy against tumor cell lines bothin vitro and in vivo.

A. Human Tumor Cell Lines

The HT-29 colon, MCF-7 breast and SKMES1 non-small cell lung human tumorcell lines were obtained from the American Type Culture Collection. TheHT-29 colon cell line was cultured in RPMI 1640 with 10% heatinactivated fetal bovine serum (HIFBS), the MCF-7 breast cell line inIscove's modified Eagles medium with 5% HIFBS plus 10-9 M insulin, andthe SKMES1 lung cell line in Eagle's minimal essential medium with 10%non-heat inactivated FBS.

B. Normal Human Marrow Cells

Normal human bone marrow (histologically negative for tumor cells) wasobtained from patients who were to have bone marrow transplants fortheir solid tumors but died before the marrow was used. Aftercentrifugation, the buffy coat was removed and cells were treated withlysis buffer and washed twice with and then resuspended in RPMI 1640with 20% HIFBS. The cells were drawn through a 25 g needle and counted.

C. Radiometric (Bactec) System

The Bactec system (Johnston Laboratories, Towson, Md.) is based on aclinical instrument which was developed to detect bacteria in bloodcultures and has been used to screen for new antineoplastic agents. Thisradiometric system is a rapid, semiautomated system which utilizes theinhibition of the conversion of ¹⁴C glucose to ¹⁴CO₂ as an index ofcytotoxicity. The Bactec machine automatically flushes out the ¹⁴CO₂into an ion chamber where the signal of the radiolabelled CO₂ is changedinto a proportional electrical signal or growth index value on a scaleof 1 to 1000. For the continuous exposure, the tumor cells or normalmarrow cells were added to 2 ml of the appropriate growth mediumcontaining 2 uC of ¹⁴C glucose plus BMOV at final concentrations of0.01, 0.1, 1, 10, 25 and 50 uM and injected into 20 ml rubber stopperedserum vials which contained a mixture of 5% CO₂ and air, and incubatedat 37° C. for 24 days. For one more hour exposure, cells and BMOV at thesame final concentrations were incubated in 15 ml polypropylene conicalsin a 37° C. water bath for one hour. The cells were then centrifuged andwashed in medium, then resuspended in 2 ml of the appropriate growthmedium containing 2 uCi of ¹⁴C glucose and injected into 20 ml rubberstoppered serum vials which contained a mixture of 5% CO₂ and air, andincubated at 37° C. for 24 days at days 6, 9 and 12 for tumor cell linesand days 6, 15 and 24 for marrow cells the vials were removed andinserted into the Bactec instrument for determination of the amount of¹⁴CO₂ produced by the cells upon metabolizing the ¹⁴C glucose. Thegrowth index values of BMOV treated cells were compared to the growthindex values of non-treated cells and the % survival compared tountreated controls was calculated.

D. Resected Tumor Studies

Seven week old, male C3H/HeJ mice were used in these studies. RIF-1(murine radiation induced fibrosarcoma) cells were cultured in alpha-MEMmedia containing 10% FBS (Gibco Canada). Cells were suspended in 1%Hanks buffered salt solution (HBSS pH 7.4) (Gibco Canada) at aconcentration of 1×10⁷ cells/ml. One hundred microliters of these cells(1×10⁶ cells) was injected into the right flank of each mouse. Thetumors were allowed to grow for 5 days (at which time the tumors rangedfrom 6 to 8 mm in diameter). At day 5 the mice were anesthetized with aKetamine:Rompom (70 mg/kg:10 mg/kg) combination (0.02 ml/g). An incisionwas made 5 mm from the tumor edge and approximately 90% of each tumorwas removed and 150 mg of molten (50° C.) PCL-BMOV or PCL alone(control) was extruded from a 500 ul syringe onto the entire surface ofthe resected tumor site. The PCL solidified within 30 seconds and thearea was closed with 5-0 prolene sutures. The mice were examined on days4, 5, 6 and 7. On each day tumors were measured (long and shortdiameters) and images taken. When the tumors reached a maximum diameterof 9 mm the mice were sacrificed and the tumor area was excised forfuture histological studies.

Results are shown in FIG. 72.

E. Tumor Inhibition Studies

The MDAY-D2 haematopoetic cell line (obtained from Dr. J Dennis, MountSinai Hospital, Toronto) was plated or grown in suspension in DMEMcontaining 5% FBS (Gibco Canada). Each mouse was injected subcutaneouslyon the posterior lateral side with 4×10⁵ cells in 100 ul of PBS. After 5days tumor growth, 150 mg of the PCL or PCL-BMOV molten paste wasimplanted in an area adjacent to the tumor site of each mouse. After 15days the mice were sacrificed, weighed and the tumors dissected andweighed. Results are shown in FIG. 71.

F. Results and Discussion

Against the normal human bone marrow cells, the one hour exposure BMOValso had very little effect, even at a concentration of 50 μM. Using acontinuous exposure, the effect of BMOV on the marrow cells was stillnot very pronounced, with sensitivity (49% survival) observed at the 50μM concentration only. Thus the BMOV compound did not appear to be verymyleosuppressive at the concentrations and exposures tested.

In this study, the antineoplastic effect of BMOV have been shown invitro against three human cancer cell lines under conditions that ensurecontinuous exposure to the drug. However, in vivo, the efficacy maydepend on the continuous exposure of the tumor cells to BMOV. The mainobjective of this study was to design and test (in vivo) a biodegradablepolymeric delivery system that might provide a continuous supply ofvanadium (BMOV form) at low concentrations.

In vivo experiments showed that single administration of PCL-BMOV pastesubcutaneously inhibited MDAY D2 tumor growth. FIG. 71 shows the datafor tumor weights from control mice (PCL-no BMOV) and mice treated with25%, 30% and 35% BMOV loaded PCL. There was a 54% inhibition of tumorgrowth for 25% BMOV loaded PCL (significant at p<0.05). The 30% and 35%BMOV loadings produced 76% and 80% inhibition of tumor growthrespectively and one of the six mice in the 35% BMOV group showedcomplete eradication of the tumor.

Interestingly, the in vivo drug release profiles showed that pasteconsisting of 25% and 30% BMOV released approximately 500 ug of BMOV perday which has previously shown efficacy. However, 35% BMOV loaded PCLpaste which was the most effective in reducing tumor growth, releasedapproximately twice this amount of drug in vitro. These data demonstratethat sustained release of low quantities of vanadium compounds will bean equally or more effective antineoplastic regime compared to a dailyvanadate administration regime.

Although, PCL-BMOV paste was equally effective in inhibiting tumorgrowth, the mice showed no signs of stress and weight loss. Increasedstress and weight loss commonly observed with daily injections of highdoses of vanadate is most likely related to toxicity induced by the highvanadate levels in the plasma immediately following administration.Using PCL-BMOV paste to provide sustained release of vanadate for longperiods may reduce large fluctuations in plasma vanadate concentrationsand prevent vanadate induced toxicity. These data are consistent withour hypothesis that a slow sustained release of BMOV is equally or moreeffective in reducing tumor growth and prevents vanadate inducedtoxicity.

Example 29 Effect of BMOV Microspheres on Tumor Growth in Mice

The objective of this study was to test the ability of BMOV-loaded PLLAmicrospheres (20%) to regress tumor growth in mice.

Twenty of twenty-four mice were injected subcutaneously with 100 ml ofMDAY-D2 cells with density of 10×10⁶/ml. On day 6 the mice were dividedinto 6 groups. Group 1, empty control, group 2, tumor control, group 3,were injected subcutaneously with 0.25 mg/100 ul BMOV, twice a day.Group 4 was injected IP with 20 mg PLLA microspheres containing 5 mg ofBMOV. Group 5 was injected IP with 10 mg of BMOV microspheres on day 6and day 9 respectively. Group 6 was injected intramuscularly with 10 mgof BMOV microspheres on day 6 and day 9. On day 16, the mice weresacrificed and tumors were dissected.

The results of these experiments are shown in Tables VI and VII below.TABLE VI Body weights of mice in control and treated groups BMOV BMOVBMOV BMOV Con- Tumor Solution beads, IP beads, IP beads, IM trol Control0.25 mg × 1 5 mg/once 2 mg × 2 2 mg × 2 1 19 23.9 18 18.8 19.2 18.2 220.2 21 17.8 19.3 18.9 22.6 3 18.4 20 18.4 21.6 17.2 20.1 4 22.2 24.117.3 19.1 22.2 Aver- 19.95 22.25 17.87 19.9 18.6 20.78 age

TABLE VII Tumor weight of control and treated groups BMOV BMOV BMOV BMOVTumor solution beads, IP beads, IP beads, Im control s.c. 5 mg/once 2 mg× 2 2 mg × 2 1 3.8 0.4 0.2 0.12 2.0 2 1.2 0.12 0.9 0.4 3.3 3 1.3 0.070.26 0.3 1.1 4 1.5 0.04 died 1.2 1.6 Average 1.95 0.15 0.45 0.50 1.6

Example 30 Controlled-Release Polymeric Drug Delivery System:Comparative Study of the In Vitro Drug Release Profiles of OrganicVanadium Complexes from Poly (ε-Caprolactone) (PCL) Thermopastes andPCL-Methoxypolyethylene Glycol (PCL-MePEG) Thermopastes

This study was conducted to investigate the encapsulation and in vitrorelease kinetics of the four organic complex forms of vanadium (BMOV,BEMOV, V5, PRC-V) in poly(ε-caprolactone) (PCL) thermopastes and/orPCL-methoxypolyethylene glycol (PCL-MePEG) thermopastes.

A. Method

Quantitative analysis (UV/VIS spectrophometric analysis) was done withdifferent concentrations of vanadium solutions to obtain the wavelengthsof peak absorbance and the calibration equations. Solubility of vanadiumwas studies in 10 mM phosphate buffered saline with albumin (PBS/ALB)(pH 7.4). 1%, 5%, 10%, and 20% (% vanadium complex in PCL) of eachorganic complex form of vanadium were encapsulated in a biodegradablepolymer PCL and/or in blends of PLC with MePEG (MW 350) to produce apolymeric drug delivery product called a “thermopaste”. In vitro releasestudies of the various forms of vanadium from thermopastes were carriedout at 37° C. in PBS/ALB with vanadium release measured by UV/VISabsorbance spectroscopy.

B. Results

Results are shown in FIGS. 73 to 77. Briefly, the peak absorbance ofBMOV, BEMOV, and V5 occurred at 276 nm and that of PRC-V was at 266 nm.The rate and extent of solubility of each form of vanadium was in theorder of V5>BMOV & BEMOV>PRC-V. In vitro release studies showed that therate of drug released from the thermopastes increased with (1)increasing drug solubility, (2) increasing drug concentration, and (3)increasing amount of MePEG incorporated into the thermopastes.

C. Conclusion

A controlled dose of vanadium may be released from PCL thermopastes byusing a different form of vanadium (as the drug), by changing the %loading of the particular form of vanadium in the PCL, or by includingMePEG into the PCL. Whereas changing the % loading of a particular formof vanadium may control the release rate in the short term, the deliveryof a controlled dose may involve the implantation of a huge (andpotentially toxic) depot of the drug (in PCL) into the animal.Therefore, the use of an alternative form of vanadium or the inclusionof MePEG into the PCL may offer an alternative method delivering acontrolled dose of vanadium.

Example 31 Encapsulation of Camptothecin into PCL Thermopaste andAnti-Angiogenesis Analysis

A. Incorporation of Camptothecin into PCL

Camptothecin was ground with a pestle and mortar to reduce the particlesize to below 5 microns. It was then mixed as a dry powder withpolycaprolactone (molecular wt. 18,000 Birmingham Polymers, AL USA). Themixture is heated to 65° C. for 5 minutes and the molten polymer/agentmixture is stirred into a smooth paste for 5 minutes. The molten pasteis then taken into a 1 ml syringe and extruded to form 3 mg pellets.These pellets were then placed onto the CAM to assess theiranti-angiogenic properties.

B. Results

Camptothecin-loaded thermopaste was effective at inhibiting angiogenesiswhen compared to control PCL pellets. At 5% drug loading, ⅘ of the CAMstested showed potent angiogenesis inhibition. In addition, at 1% and0.25% loading, ⅔ and ¾ of the CAMs showed angiogenesis inhibitionrespectively. Therefore, it is evident from these results thatcamptothecin was sufficiently released from the PCL thermopaste and ithas therapeutic anti-angiogenic efficacy.

Example 32 Manufacture of S-Phosphonate-Loaded Thermopaste

In this study, we demonstrated that s-phosphonate, an ether lipid withantineoplastic activity, was successfully incorporated into PCLthermopaste and showed efficacy in the CAM assay.

A. Manufacture of S-phosphonate Loaded Paste

Polycaprolactone (Birmingham Polymers, Birmingham, Ala.) ands-phosphonate were levigated in the appropriate proportions at 55° C.for 2 minutes. The molten mixture was then pipetted into 3 mgsemi-spherical pellets and allowed to set at 4° C.

B. CAM Bioassay

Fertilized, domestic chick embryos (Fitzsimmons Consulting & ResearchServices Ltd., B.C.) were incubated for 4 days and then windowed.Briefly, a small hole (measuring approximately 2 cm in diameter) wasformed by removing the shell and inner shell membrane from the blunt endof the egg (air space site) and then the exposed area was sealed withsterilized Parafilm wax. The egg was then placed into an incubator at37° C. for an additional 2 days with the window upright. On day 6 ofincubation, 3 mg pellets of s-phosphonate-loaded polymer or control (nodrug) polymer was placed on the surface of the growing CAM vessels.After a 2 day exposure (day 8 of incubation), the vasculature wasexamined using a stereomicroscope fitted with a Contax camera system. Toincrease the contrast of the vessels and to mask any backgroundinformation, the CAM was injected with 1 ml of intralipid solution(Abbott Laboratories) prior to imaging.

C. Results

PCL pellets loaded with s-phosphonate at 0%, 1%, 2%, 4% and 8% induced adose-dependent anti-angiogenic reaction in the CAM. The anti-angiogenicreaction is characterized by the absence of blood vessels in the regiondirectly below the s-phosphonate loaded pellet. The normal growth of thedense capillary network seen in the control CAM has clearly beeninhibited in the s-phosphonate treated CAM's. At higher concentrations(4% and 8%), the treated CAMs were structurally altered in the vicinityof the drug/polymer pellet. This alteration included a pronouncedthickening of the CAM immediately adjacent to the s-phosphonate/polymerpellet and membrane thinning subjacent to the pellet. In all of the CAMstreated with s-phosphonate, an avascular zone was apparent after a twoday exposure; this was defined as an area devoid of a capillary networkmeasuring approximately 3 mm² in area.

Example 33 Encapsulation of Tyrosine Kinase Inhibitors into PCLThermopaste and Analysis Using the Cam Assay

Tyrosine kinase inhibitors were ground with a pestle and mortar toreduce the particle size to below 5 microns. They were then mixed as adry powder with polycaprolactone (molecular wt. 18000 BirminghamPolymers, AL USA). The mixture is heated to 65° C. for 5 minutes and themolten polymer/agent mixture is stirred into a smooth paste for 5minutes. The molten paste is then taken into a 1 ml syringe, andextruded to form 3 mg pellets. These pellets were then tested in the CAMassay. The tyrosine kinase inhibitors that were tested in the CAM assayinclude, lavendustine-c, erbstatin, herbimysin, and genistein. Whencomparing the anti-angiogenic inhibition effects of these agents,herbimysin (2% in PCL) was the most potent inducing an avascular zone in4/4 of the CAMs tested.

Example 34 Encapsulation of Vinca Alkaloids into PCL Thermopaste andAnalysis using the Cam Assay

A. Incorporation of Inhibitors into PCL Thermopaste

Vinca alkaloids (vinblastine and vincristine) were ground with a pestleand mortar to reduce the particle size to below 5 microns. They werethen mixed as a dry powder with polycaprolactone (molecular wt. 18000Birmingham Polymers, AL USA). The mixture is heated to 65° C. for 5minutes and the molten polymer/agent mixture is stirred into a smoothpaste for 5 minutes. The molten paste is then taken into a 1 ml syringeand extruded to form 3 mg pellets. These pellets were then tested in theCAM assay.

B. Results

When testing the formulations on the CAM, it was evident that the agentswere being released from the PCL pellet in sufficient amounts to inducea biological effect. Both vinblastine and vincristine inducedanti-angiogenic effects in the CAM assay when compared to control PCLthermopaste pellets.

At concentrations of 0.5% and 0.1% drug loading, vincristine inducedangiogenesiss inhibition in all of the CAMs tested. When concentrationsexceeding 2% were tested, toxic drug levels were achieved and unexpectedembryonic death occurred.

Vinblastine was also effective in inhibiting angiogenesis on the CAM atconcentrations of 0.25%, 0.5% and 1%. However, at concentrationsexceeding 2%, vinblastine was also toxic to the embryo.

Example 35 Bioadhesive Microspheres

A. Preparation of Bioadhesive Microspheres

Microspheres were made from 100 k g/mol PLLA with a particle diameterrange of 10-60 μm. The microspheres were incubated in a sodium hydroxidesolution to produce carboxylic acid groups on the surface by hydrolysisof the polyester. The reaction was characterized with respect to sodiumhydroxide concentration and incubation time by measuring surface charge.The reaction reached completion after 45 minutes of incubation in 0.1 Msodium hydroxide. Following base treatment, the microspheres were coatedwith dimethylaminoproylcarbodiimide (DEC), a cross-linking agent bysuspending the microspheres in an alcoholic solution of DEC and allowingthe mixture to dry into a dispersible powder. The weight ratio ofmicrospheres to DEC was 9:1. After the microspheres ere dried, they weredispersed with stirring into a 2% w/v solution of poly (acrylic acid)and the DEC allowed to react with PAA to produce a water insolublenetwork of cross-linked PAA on the microspheres surface. Scanningelectron microscopy was used to confirm the presence of PAA on thesurface of the microspheres.

Differential scanning calorimetry of the microspheres before and aftertreatment with base revealed that no changes in bulk thermal properties(Tg, melting, and degree of crystallinity) were observed by scanningelectron microscopy.

B. In Vitro Paclitaxel Release Rates

Paclitaxel loaded microspheres, (10% and 30% w/w loadings) with the sameparticle diameter size range were manufactured and in vitro releaseprofiles for 10 days release in phosphate buffered saline. Release wasproportional to drug loading, with 400 μg of Paclitaxel released from 5mg of 30% loaded microspheres in 10 days and 150 μg released from 10%loaded microspheres in the same period. The efficiency of encapsulationwas about 80%. The Paclitaxel loaded microspheres were incubated in 0.1M sodium hydroxide for 45 minutes and the zeta potential measured beforeand after incubation in sodium hydroxide. The surface charge ofPaclitaxel loaded microspheres was lower than microspheres with noPaclitaxel both before and after treatment with base.

C. Preparation and In Vitro Evaluation of PLLA Coated with eitherPoly-Lysine or Fibronectin.

PLLA microspheres were prepared containing 1% sudan black (to color themicrospheres). These spheres were suspended in a 2% (w/volume) solutionof either poly-lysine (Sigma chemicals—Hydrobromell form) or Fibronectin(Sigma) for 10 minutes. The microspheres were wasted in buffer once andplaced on the inner surface of freshly prepared bladders from rats. Thebladder were left for 10 minutes then washed three times in buffer.Residual (bound) microspheres were present on the bladder wall after theprocess therefore showing bioadhesion had occurred (FIGS. 78A and 78B)for both Fibronectin and poly-1-lysine coated microspheres.

Example 36 Synthesis of Poly(N-Isopropylacrylamide)

Polyacrylamide and its derivatives may be readily synthesized throughfree radical polymerization and irradiation induced polymerization. Thefollowing is an example of synthesizing poly(N-isopropylacrylamide),wherein the monomer N-isopropylacrylamide is purified byrecrystallization from an organic solvent such as hexane and toluene.

Briefly, N-isopropylacrylamide is dissolved in toluene at a temperaturesuch as 60° C. This solution is cooled down to a lower temperature (e.g.4° C.) to allow the recrystallization of the monomer. The monomer isthen collected by filtration and dried under vacuum. For synthesis, thepurified monomer (20 g) is dissolved in distilled water (180 ml) in around bottomed glass flask. The solution is purged with nitrogen for 30minutes to replace dissolved oxygen. The temperature is then raised to65° C. and a small amount (0.1 g) of initiator such as ammoniumpersulfate and 2,2′-azobis-isobutyronitrile is added to start thepolymerization. The polymerization is completed within 10 hours and isunder the protection of nitrogen and stirring. Finally,poly(N-isopropylacrylamide) is precipitated by adding ethanol. Thepolymer is dried and stored.

Example 37 Synthesis of Poly(Acrylic Acid) Derivatives

Poly(acrylic acid) and its derivatives can be synthesized through freeradical polymerization and irradiation induced polymerization. Thefollowing is an example of synthesizing poly(acrylic acid), whereinmonomer acrylic acid is purified by distillation (e.g., 40° C.) under areduced pressure (e.g., 10 mmHg).

Briefly, the purified monomer (20 g) is dissolved in dioxane (180 ml) ina round bottomed glass flask. The solution is purged with nitrogen for30 minutes to replace the dissolved oxygen. The temperature is thenraised to 65° C. and a small amount (0.1 g) of initiator2,2′-azobis-isobutyronitrile is added to start the polymerization. Thepolymerization is completed within 24 hours and is under the protectionof nitrogen and stirring. Finally, poly(acrylic acid) is precipitated byadding n-hexane. The polymer is dried and stored.

Example 38 Preparation of Micellular Paclitaxel

A. Synthesis of Diblock Copolymer of PDLLA-MePEG

Monomers of methoxy polyethylene glycol (e.g., M.W. 2000, 150 g) andDL-lactide (100 g) are added to a round bottomed flask and thetemperature is raised to 130-150° C. After the melting of the monomers,0.6 g catalyst stannous octoate is added. The polymerization is finishedwithin 0 hours. The reaction is under protection of N₂ and withstirring.

B. Preparation of Micellar Paclitaxel

The PDLLA-MePEG copolymer is dissolved in acetonitrile or 50:50ethanol:acetone (polymer conc.<40%). The polymer solution is centrifuged(14000 rpm) for 5 min. The supernatant (insoluble polymer discarded) istransferred to a glass test tube. Paclitaxel is dissolved inacetonitrile or 50:50 ethanol:acetone and is added to the purifiedpolymer solution. After vortex mixing, the solvent is evaporated at 60°C. under a stream of nitrogen (normally takes 2 hours for a typical 40mg paclitaxel batch). The residual solvent can be removed by applyingvacuum and heat. The matrix is heated at about 60° C. until it becomes atransparent gel. Then a certain volume of water (>4 times of matrixweight) is added to the matrix. This is followed immediately by vortexmixing until the solubilization of the paclitaxel/polymer matrix.

C. Preparation of Delivery Systems of Micellar Paclitaxel/ThermogellingPolymers

A thermogelling polymer such as poly(N-isopropylacrylamide), isdissolved in distilled water. Separately, water is added to a micellarpaclitaxel/polymer matrix, e.g., 10% paclitaxel loaded PDLLA-MePEG2000-40/60, to form a micellar paclitaxel solution. Both solutions arecooled (e.g., 4° C.) and mixed to form the thermogelling deliverysystem.

Example 39 Perivascular Administration of Paclitaxel

WISTAR rats weighing 250-300 g are anesthetized by the intramuscularinjection of Innovar (0.33 ml/kg). Once sedated they are then placedunder Halothane anesthesia. After general anesthesia is established, furover the neck region is shaved, the skin clamped and swabbed withbetadine. A vertical incision is made over the left carotid artery andthe external carotid artery exposed. Two ligatures are placed around theexternal carotid artery and a transverse arteriotomy is made. A number 2FRENCH FOGART balloon catheter is then introduced into the carotidartery and passed into the left common carotid artery and the balloon isinflated with saline. The catheter is passed up and down the carotidartery three times. The catheter is then removed and the ligature istied off on the left external carotid artery.

Paclitaxel (33%) in ethelyne vinyl acetate (EVA) is then injected in acircumferential fashion around the common carotid artery in ten rats.EVA alone is injected around the common carotid artery in ten additionalrats. Five rats from each group are sacrificed at 14 days and the finalfive at 28 days. The rats are observed for weight loss or other signs ofsystemic illness. After 14 or 28 days the animals are anesthetized andthe left carotid artery is exposed in the manner of the initialexperiment. The carotid artery is isolated, fixed at 10% bufferedformaldehyde and examined for histology.

Example 40 Treatment of Artherosclerosis

A. Atherosclerosis

Atherosclerotic lesions are created in New Zealand white rabbits by dietonly. Briefly, New Zealand white rabbits weighing approximately 1.6 kgare placed on a powdered chow supplemented by 0.25% cholesterol byweight. Total plasma cholesterol is measured on a weekly basis by takingsamples from a marginal ear vein after an injection of Innovar (0.1ml/kg) to dilate blood vessels. Samples are mixed with EDTA to achieve a0.15% concentration in the sample and placed on ice until separation ofplasma by low speed centrifugation.

One week after initiation of the full cholesterol diet, the animals arerandomized into 3 groups of 10. After anaesthetic induction withKetamine 35 mg/kg and Xylazine 7 mg/kg, and then general anesthesia viaintubation, the fur is shaved and the skin sterilized over the abdomen.A laparotomy is performed and the abdominal aorta isolated. Using a 22 gneedle, ethylene vinyl acetate paste, ethylene vinyl acetate pastecontaining 5% paclitaxel, or ethylene vinyl acetate paste containing 33%paclitaxel is placed in a circumferential manner around the proximalhalf of the infrarenal abdominal aorta. The distal half of the aortaextending to the aortic bifurcation is not treated. In 10 controlrabbits, the infrarenal abdominal aorta is isolated, but nothing isinjected around it.

The atherogenic chow is continued for 24 weeks. At that time, theanimals are anesthetized with an injection of Ketamine (350 mg/kg) andxylazine (7 mg/kg) intramuscularly and then sacrificed with anintravenous overdose of Euthanol (240 mg/ml; 2 ml/4.5 kg). The animalsare then perfusion fixed at 100 mm mercury via the left ventricle byperfusing Hanks' balanced salt solution with 0.15 mmol/litreN-2-hydroxyethylpaparazine-N′-2-ethanesulfonic acid (ph 7.4) containingHeparin (1 IU/ml) for ten minutes followed by dilute Karnovsky'sfixative for 15 minutes. The thoracic and abdominal aorta and iliacarteries are removed en bloc and are placed in a similar solution for afurther 30 minutes.

Serial thin sections are then performed through the thoracic aorta andparticularly through the infrarenal abdominal aorta. Movat, H&E, andMasson stains are performed and histologic analysis made to examined thedegree of luminal compromise, the degree of atherosclerotic lesiondevelopment, and any perilumenl reaction to the circumferential arterialmedications.

Example 41 Treatment of Restenosis

WISTAR rats weighing 250-300 g are anesthetized by the intramuscularinjection of innovar (0.33 ml/kg). Once they are sedated they are placedunder Halothane anesthesia. After general anesthesia is established, thefur over the neck region is shaved and the skin cleansed with betadine.A vertical incision is made over the left carotid artery and theexternal carotid artery exposed. Two ligatures are placed around theexternal carotid artery and a transverse arteriotomy is made betweenthem. A 2 Fr Fogarty balloon catheter is introduced into the externalcarotid artery and passed into the left common carotid artery and theballoon is inflated with saline. The catheter is passed up and down thecarotid artery three times to denude the endothelium. The catheter isremoved and the ligatures tied off on the left external carotid artery.

The animals are randomized into groups of 5. Subgroups of 5 rats arecontrol, carrier polymer alone, carrier polymer plus 1, 5, 10, 20, and33% paclitaxel is delivered. There are two carrier polymers to beinvestigated; EVA and EVA/PLA blend. The polymer mixture is placed in acircumferential manner around the carotid artery. The wound is thenclosed. Rats in each group are sacrificed at 14 and 28 days. In theinterim, the rats are observed for weight loss or other signs ofsystemic. After 14 or 28 days, the animals are sacrificed by initialsedation with intramuscular Innovar (0.33 ml/kg). The arteries are thenexamined for histology.

Example 42 Intimal Hyperplasia Causing Graft Stenosis

A. Animal Studies

General anaesthesia is induced into domestic swine. The neck region isshaved and the skin sterilized with cleansing solution. Verticalincisions are made on each side of the neck and the carotid artery isexposed and 8 mm PTFE graft is inserted by 2 end to side anastomoses,the proximal anastomosis on the common carotid artery and the distalanastomosis on the internal carotid artery bilaterally. The interveningbypassed artery is ligated. The animals are randomized into groups of 10pigs receiving carrier polymer alone, 10 pigs receiving carrier polymerplus 5% paclitaxel, and 10 pigs receiving carrier polymer plus 33%paclitaxel adjacent to each surgical created anastamosis on the leftside only. The right sided grafts will serve as a control in each pig.The wounds are closed and the pigs recovered.

A second group of pigs are studied. The grafts are created in a similarmanner. No vasoactive agent is placed next to the anastamotic sites atthe time of operation. The animals are recovered. Two weeks after thegraft has been performed, a second general anaesthetic is administeredand the left carotid artery is reexplored. Adjacent to the proximal anddistal anastamoses, 10 pigs each receive carrier alone, carrier polymerplus 5% paclitaxel and carrier polymer plus 33% paclitaxel in acircumferential manner adjacent to both proximal and distal anastamoses.The wounds are closed and the pigs recovered. Opposite the right sidedgraft serves as a control.

At 3 months, all pigs undergo general anesthetic. A cutdown is made onthe femoral artery and a pigtail catheter is inserted in the ascendingthoracic aorta under fluoroscopic guidance. Arch injection with imagingof the carotid vasculature is performed. Specifically, the degree ofstenoses of the proximal and distal grafts and the artery immediatelydistal to the distal anastamosis of the graft is measured and the %stenosis calculated. If necessary, selective injections of the commoncarotid arteries are performed.

Five pigs in each group are sacrificed. The animals are then perfusionfixed at 100 mm mercury via the left ventricle by perfusing Hanks'balanced salt solution with 0.15 mmol/litreN-2-hydroxyethylpaparazine-N′-2-ethanesulfonic acid (ph 7.4) containingHeparin (1 IU/ml) for ten minutes followed be dilute Karnovsky'sfixative for 15 minutes. The thoracic and abdominal aorta and carotidarteries are removed en bloc and are placed in a similar solution for afurther 30 minutes.

Histological sections through the carotid artery immediately proximal tothe proximal anastamosis, at the proximal anastamosis, at the distalanastamosis and the carotid artery immediately distal to the distalanastamosis are made. The sections are stained with Movat and H&E andMasson stains. Histologic analysis of intimal and advantitial reactionas well as perivascular reaction are noted. Morphometric analysis withdegree of luminal narrowing is calculated.

The remaining pigs are studied at 6 months and a similar angiographysacrifice procedures is performed.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A method for treating or preventing a disease associated with a bodypassageway, comprising delivering to an external portion of the bodypassageway a therapeutic agent or composition thereof, wherein thetherapeutic agent or composition thereof is delivered from a thread. 2.The method of claim 1, comprising delivering the therapeutic agent orcomposition thereof via the adventitia to smooth muscle cells of thebody passageway.
 3. The method of claim 1 wherein the therapeutic agentis an anti-angiogenic factor.
 4. The method of claim 1 wherein thetherapeutic agent is a compound which disrupts microtubule function. 5.The method of claim 4 wherein the compound which disrupts microtubulefunction is paclitaxel, or an analogue, derivative, or conjugatethereof.
 6. The method of claim 5 wherein the compound which disruptsmicrotubule function is paclitaxel.
 7. The method of claim 1 wherein thecomposition comprises the therapeutic agent and a polymer.
 8. The methodof claim 7 wherein the composition is biodegradable.
 9. The method ofclaim 7 wherein the composition is non-biodegradable.
 10. The method ofclaim 7 wherein the polymer is biodegradable.
 11. The method of claim 7wherein the polymer is non-biodegradable.
 12. The method of claim 7wherein the polymer comprises poly(ethylene-vinyl acetate).
 13. Themethod of claim 7 wherein the polymer comprises a polyester.
 14. Themethod of claim 7 wherein the composition comprises a polymer whereinthe polymer is poly (D,L-lactide), poly (D,L-lactic acid), poly(L-lactic acid), a copolymer formed from lactic acid and glycolic acid,a copolymer of poly (lactic acid) and poly (caprolactone), apolyanhydride, poly (caprolactone), or a copolymer of poly(lactic acid)and poly(ethylene glycol).
 15. The method of claim 7 wherein the polymercomprises poly(D,L-lactide-co-glycolide).
 16. The method of claim 7wherein the polymer is in the form of a coating.
 17. The method of claim1 wherein the body passageway is the esophagus, the stomach, theduodenum, the small intestine, the large intestine, biliary tracts, theureter, the bladder, the urethra, lacrimal ducts, the trachea, bronchi,bronchiole, nasal airways, eustachian tubes, the external auditorycanal, fallopian tubes, oral cavities, the uterus, vagina or otherpassageways of the female reproductive tract, the vas deferens or otherpassageways of the male reproductive tract, or the ventricular system ofthe brain or the spinal cord.
 18. The method of claim 1 wherein the bodypassageway is an artery, a vein, or a capillary.
 19. The method of claim1 wherein the disease is stenosis or restenosis.